Non-affinity purification of proteins

ABSTRACT

The present invention relates to a method for protein purification that involves the combination of non-affinity chromatography with HPTFF.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to protein purification. In particular,the invention relates to a method for purifying proteins (such asantibodies and antibody-like molecules, e.g. immunoadhesins) from acomposition comprising the polypeptide and at least one impurity withoutthe use of affinity chromatography.

2. Description of the Related Art

The large-scale, economic purification of proteins is increasingly animportant problem for the biotechnology industry. Generally, proteinsare produced by cell culture, using either mammalian or bacterial celllines engineered to produce the protein of interest by insertion of arecombinant plasmid containing the gene for that protein. Since the celllines used are living organisms, they must be fed with a complex growthmedium, containing sugars, amino acids, and growth factors, usuallysupplied from preparations of animal serum. Separation of the desiredprotein from the mixture of compounds fed to the cells and from theby-products of the cells themselves to a purity sufficient for use as ahuman therapeutic poses a formidable challenge.

Procedures for purification of proteins from cell debris initiallydepend on the site of expression of the protein. Some proteins arecaused to be secreted directly from the cell into the surrounding growthmedia; others are made intracellularly. For the latter proteins, thefirst step of a purification process involves lysis of the cell, whichcan be done by a variety of methods, including mechanical shear, osmoticshock, or enzymatic treatments. Such disruption releases the entirecontents of the cell into the homogenate, and in addition producessubcellular fragments that are difficult to remove due to their smallsize. These are generally removed by centrifugation or by filtration.The same problem arises, although on a smaller scale, with directlysecreted proteins due to the natural death of cells and release ofintracellular host cell proteins in the course of the protein productionrun.

Once a solution containing the protein of interest is obtained, itsseparation from the other proteins produced by the cell is usuallyattempted using a combination of different chromatography techniques.These techniques separate mixtures of proteins on the basis of theircharge, degree of hydrophobicity, or size. Several differentchromatography resins are available for each of these techniques,allowing accurate tailoring of the purification scheme to the particularprotein involved. The essence of each of these separation methods isthat proteins can be caused either to move at different rates down along column, achieving a physical separation that increases as they passfurther down the column, or to adhere selectively to the separationmedium, being then differentially eluted by different solvents. In somecases, the desired protein is separated from impurities when theimpurities specifically adhere to the column, and the protein ofinterest does not, that is, the protein of interest is present in the“flow-through.”

Ion-exchange chromatography, named for the exchangeable counterion, is aprocedure applicable to purification of ionizable molecules. Ionizedmolecules are separated on the basis of the non-specific electrostaticinteraction of their charged groups with oppositely charged moleculesattached to the solid phase support matrix, thereby retarding thoseionized molecules that interact more strongly with solid phase. The netcharge of each type of ionized molecule, and its affinity for thematrix, varies according to the number of charged groups, the charge ofeach group, and the nature of the molecules competing for interactionwith the charged solid phase matrix. These differences result inresolution of various molecule types by ion-exchange chromatography. Intypical protein purification using ion exchange chromatography, amixture of many proteins derived from a host cell, such as in mammaliancell culture, is applied to an ion-exchange column. After non-bindingmolecules are washed away, conditions are adjusted, such as by changingpH, counter ion concentration and the like in step- or gradient-mode, torelease from the solid phase a non-specifically retained or retardedionized protein of interest and separating it from proteins havingdifferent charge characteristics. Anion exchange chromatography involvescompetition of an anionic molecule of interest with the negative counterion for interaction with a positively charged molecule attached to thesolid phase matrix at the pH and under the conditions of a particularseparation process. By contrast, cation exchange chromatography involvescompetition of a cationic molecule of interest with the positive counterion for a negatively charged molecule attached to the solid phase matrixat the pH and under the conditions of a particular separation process.Mixed mode ion exchange chromatography involves the use of a combinationof cation and anion exchange chromatographic media in the same step. Inparticular, “mixed-mode” refers to a solid phase support matrix to whichis covalently attached a mixture of cation exchange, anion exchange, andhydrophobic interaction moieties. A commercially availablerepresentative of mixed-mode ion exchange chromatographic columns isABx™, the use of which is described in the Examples.

Hydroxyapatite chromatography of proteins involves the non-specificinteraction of the charged amino or carboxylate groups of a protein withoppositely charged groups on the hydroxyapatite, where the net charge ofthe hydroxyapatite and protein are controlled by the pH of the buffer.Elution is accomplished by displacing the non-specificprotein-hydroxyapatite pairing with ions such as Ca²⁺ or Mg²⁺.Negatively charged protein groups are displaced by negatively chargedcompounds, such as phosphates, thereby eluting a net-negatively chargedprotein.

Hydrophobic interaction chromatography (HIC) is useful for thepurification and separation of molecules, such as proteins, based ondifferences in their surface hydrophobicity. Hydrophobic groups of aprotein interact non-specifically with hydrophobic groups coupled to thechromatography matrix. Differences in the number and nature of proteinsurface hydrophobic groups results in differential retardation ofproteins on an HIC column and, as a result, separation of proteins in amixture of proteins.

Hydrophobic charge induction (HCI) chromatography is useful for theseparation of biological molecules, such as proteins, based on thepH-dependent behavior of ionizable, dual-mode ligands (Boschetti, E. etal., Genetic Engineering News 20(13) (2000)). At neutral pH, the ligandis uncharged and binds a protein of interest via mild non-specifichydrophobic interaction. As pH is reduced during a buffer gradient, theligand becomes positively charged and hydrophobic binding is disruptedby electrostatic charge repulsion (Boschetti, E. (2000), supra). Thegentle conditions used in HCI reduces the risk of protein denaturationand antibody aggregation.

Affinity chromatography, which exploits a specific structurallydependent (i.e., spatially complementary) interaction between theprotein to be purified and an immobilized capture agent, is a standardpurification option for some proteins, such as antibodies. Protein A,for example, is a useful adsorbent for affinity chromatography ofproteins, such as antibodies, which contain an Fc region. Protein A is a41 kD cell wall protein from Staphylococcus aureas which binds with ahigh affinity (about 10⁻⁸M to human IgG) to the Fc region of antibodies.Despite its common use, affinity chromatography is costly, particularlyat the industrial scale necessary to purify therapeutic proteins.

High-performance tangential-flow filtration (HPTFF) is a membranetechnology useful for the separation of protein mixtures without limitto their relative size (Zydney, A. L. and van Reis, R., High-PerformanceTangential-Flow Filtration, ch. 10, in Membrane Separations inBiotechnology, 2d ed., William K. Wang, ed., Marcel Dekker, Inc., NY,N.Y. (2001), pp. 277-298; van Reis, R. et al. Biotechnol. Bioeng.56:71-82 (1997); and van Reis, R., U.S. Pat. No. 5,256,694, U.S. Pat.No. 5,490,937, and U.S. Pat. No. 6,054,051, the contents of which arehereby incorporated by reference in their entirety). HPTFF can be usedthroughout the downstream purification process to remove specificimpurities (such as proteins, DNA, or endotoxins), clear viruses, and/oreliminate protein oligomers or degradation products. HPTFF is uniqueamong available separation technologies in that it can effectsimultaneous purification, concentration, and buffer exchange, allowingseveral different separations steps to be combined into a singlescalable unit operation.

Despite these advanced chromatography and filtration methods, affinitychromatography is often employed as a capture step to meet the purity,yield, and throughput requirements for pharmaceutical antibodypurification. The high cost and instability of affinity media, however,increases the ultimate cost of antibody therapeutics, particularly thoserequiring high doses and/or chronic administration. In addition,adequate purity often is not achieved unless several purification stepsare combined, thereby further increasing cost and reducing productyield. Antibodies account for an increasingly large percentage oftherapeutic products on the market and in development in the UnitedStates for the treatment of, for example, cancer, autoimmune disease,infectious disease, cardiovascular disease, and transplant rejection(Stratan, F. et al., Monoclonal Antibodies—Coming of Age, 1 (2001), andBooth, M. et al., Monoclonal Antibodies: Targeting the Issues, 1(2001)). Consequently, there is a need for processes that purify proteintherapeutics or other polypeptide compounds using fewer steps andwithout the need for a costly affinity step.

SUMMARY OF THE INVENTION

The present invention relates to the surprising finding that anon-affinity chromatographic purification process in combination withHPTFF is capable of purifying a target protein, such as an antibody oran antibody-like molecule, from a mixture containing host cell proteinssuch that host cell protein impurities are present in the final purifiedtarget protein in an amount less than 100 parts per million (ppm).

In one aspect, the invention concerns a method for purifying a targetprotein from a mixture containing a host cell protein and optionallyfurther impurities, comprising two non-affinity purification stepsfollowed by high-performance tangential-flow filtration (HPTFF), in theabsence of an affinity chromatography step, wherein such method producesa purified target protein containing less than 100 parts per million(ppm) of the host cell protein, alternatively less than 90 ppm, lessthan 80 pmm, less than 70 ppm, less than 60 ppm, less than 50 ppm, lessthan 40 ppm, less than 30 ppm, less than 20 ppm, less than 10 ppm, lessthan 5 ppm, or less than 3 ppm.

In a particular embodiment, the first and second non-affinitychromatography purification steps are different and are selected fromthe group consisting of ion exchange chromatography and hydrophobicinteraction chromatography. For example, the ion exchange chromatographystep may be cation exchange chromatography, anion exchangechromatography and/or mixed mode ion exchange chromatography. In apreferred embodiment, the first and second non-affinity purificationsteps are cation exchange chromatography and anion exchangechromatography, in either order. In another preferred embodiment, thefirst non-affinity purification step is cation exchange chromatographyand said second non-affinity purification step is anion exchangechromatography. In yet another preferred embodiment, the method of theinvention consists of two non-affinity chromatography purification stepsfollowed by HPTFF and followed by an isolation step, to the exclusion ofany other purification steps.

The target protein to be purified can be any protein, in particularrecombinant protein produced in any host cell, including but not limitedto, Chinese hamster ovary (CHO) cells. Optimal target proteins areantibodies, immunoadhesins and other antibody-like molecules, such asfusion proteins including a C_(H)2/C_(H)3 region.

In another aspect, the invention concerns a method for purifying atarget protein from a mixture containing a host cell protein andoptionally further impurities, comprising one non-affinitychromatography purification step followed by high-performancetangential-flow filtration (HPTFF), in the absence of an affinitychromatography step, wherein such method produces a purified targetprotein containing less than 100 parts per million (ppm) of the hostcell protein, alternatively less than 90 ppm, less than 80 pmm, lessthan 70 ppm, less than 60 ppm, less than 50 ppm, less than 40 ppm, lessthan 30 ppm, less than 20 ppm, less than 10 ppm, less than 5 ppm, orless than 3 ppm

These and other non-limiting embodiments of the present invention arereadily understood by one of ordinary skill in the art upon reading thedisclosure and claims provided herein. It is understood that thisinvention is not limited to the particular compositions of matter andprocesses described, as such compounds and methods may, of course, vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of the filtration set-up for HPTFFexperiments.

FIG. 2 shows a silver-stained (Zaxis 10-20%) polyacrylamide gel,containing samples that were taken at different intervals during thepurification of anti-HER2 rhuMAb and were subjected to SDS-PAGEanalysis. The arrows indicating 160 kD, 50 kD, and 25 kD point to thefull length antibody, the heavy chain, and the light chain,respectively. Other bands are anti-HER2 rhuMAb fragments. Samplesincluded a molecular weight standard (lane 1), reference rhuMAb obtainedusing an affinity-purification process (lanes 2 and 7), a sample ofrhuMAb harvested cell culture fluid (HCCF) (lane 3) prior topurification, and samples of material recovered, after S chromatography(lane 4), after Q chromatography (lane 5), after additional HPTFF (lanes6 and 12), after HPTFF Experiment 1 using CRC100+ (lane 9), after HPTFFExperiment 1 using CRC300+ (lane 10), and after HPTFF Experiment 2 (lane11).

FIGS. 3A and 3B. FIG. 3A is the amino acid sequence of the anti-HER2rhuMAb light chain; FIG. 3B is the amino acid sequence of the anti-HER2rhuMAb heavy chain.

FIGS. 4A and 4B. FIG. 4A is the amino acid sequence of the anti-CD11arhuMAb light chain; FIG. 4B is the amino acid sequence of the anti-CD11arhuMAb heavy chain.

FIG. 5 shows a silver-stained SDS-PAGE gel containing samples that weretaken at different points during the purification of anti-CD40recombinant human monoclonal antibody (rhuMAb)

DETAILED DESCRIPTION OF EMBODIMENTS

A. Definitions

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they retain,or are modified to comprise, a ligand-specific binding domain. Theantibody herein is directed against an “antigen” of interest.Preferably, the antigen is a biologically important polypeptide andadministration of the antibody to a mammal suffering from a disease ordisorder can result in a therapeutic benefit in that mammal. However,antibodies directed against nonpolypeptide antigens (such astumor-associated glycolipid antigens; see U.S. Pat. No. 5,091,178) arealso contemplated. Where the antigen is a polypeptide, it may be atransmembrane molecule (e.g. receptor) or ligand such as a growthfactor. Exemplary antigens include those polypeptides discussed above.Preferred molecular targets for antibodies encompassed by the presentinvention include CD polypeptides such as CD3, CD4, CD8, CD19, CD20,CD34, and CD40; members of the HER receptor family such as the EGFreceptor, HER2, HER3 or HER4 receptor; cell adhesion molecules such asLFA-1, Mac1, p150,95, VLA-4, ICAM-1, VCAM and av/b3 integrin includingeither a or b subunits thereof (e.g. anti-CD11a, anti-CD18 or anti-CD11bantibodies); growth factors such as VEGF; IgE; blood group antigens;flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4;polypeptide C etc. Soluble antigens or fragments thereof, optionallyconjugated to other molecules, can be used as immunogens for generatingantibodies. For transmembrane molecules, such as receptors, fragments ofthese (e.g. the extracellular domain of a receptor) can be used as theimmunogen. Alternatively, cells expressing the transmembrane moleculecan be used as the immunogen. Such cells can be derived from a naturalsource (e.g. cancer cell lines) or may be cells which have beentransformed by recombinant techniques to express the transmembranemolecule.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;single-chain antibody molecules; diabodies; linear antibodies; andmultispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352:624-628 (1991) and Marks et al, J. Mol.Biol. 222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (i.e. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “Framework”or “FR” residues are those variable domain residues other than thehypervariable region residues as herein defined.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, Fv framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the “binding domain” of a heterologous “adhesin”protein (e.g. a receptor, ligand or enzyme) with the effector functionsof an immunoglobulin constant domain. Structurally, the immunoadhesinscomprise a fusion of the adhesin amino acid sequence with the desiredbinding specificity which is other than the antigen recognition andbinding site (antigen combining site) of an antibody (i.e. is“heterologous”) and an immunoglobulin constant domain sequence. Theimmunoglobulin constant domain sequence in the immunoadhesin ispreferably derived from γ1, γ2, or γ4 heavy chains since immunoadhesinscomprising these regions can be purified by Protein A chromatography(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)).

The term “ligand binding domain” as used herein refers to any nativecell-surface receptor or any region or derivative thereof retaining atleast a qualitative ligand binding of a corresponding native receptor.In a specific embodiment, the receptor is from a cell-surfacepolypeptide having an extracellular domain which is homologous to amember of the immunoglobulin super gene family. Other receptors, whichare not members of the immunoglobulin super gene family but arenonetheless specifically covered by this definition, are receptors forcytokines, and in particular receptors with tyrosine kinase activity(receptor tyrosine kinases), members of the hematopoietin and nervegrowth factor receptor superfamilies, and cell adhesion molecules, e.g.(E-, L- and P-) selectins.

The term “receptor binding domain” is used to designate any nativeligand for a receptor, including cell adhesion molecules, or any regionor derivative of such native ligand retaining at least a qualitativereceptor binding ability of a corresponding native ligand. Thisdefinition, among others, specifically includes binding sequences fromligands for the above-mentioned receptors.

An “antibody-immunoadhesin chimera” comprises a molecule which combinesat least one binding domain of an antibody (as herein defined) with atleast one immunoadhesin (as defined in this application). Exemplaryantibody-immunoadhesin chimeras are the bispecific CD4-IgG chimerasdescribed in Berg et al., PNAS (USA) 88:4723-4727 (1991) and Chamow etal., J. Immunol. 153:4268 (1994).

Unless indicated otherwise, the term “HER2” when used herein refers tohuman HER2 protein and “HER2” refers to human HER2 gene. The human HER2gene and HER2 protein are described in Semba et al., PNAS (USA)82:6497-6501 (1985) and Yamamoto et al. Nature 319:230-234 (1986)(Genebank accession number X03363), for example.

“Trastuzumab,” “HERCEPTIN®,” “anti-HER2 rhuMAb,” and “HER2” are usedinterchangeably herein to refer to a humanized anti-HER2 antibodycomprising the light chain amino acid sequence of SEQ ID NO:1 and theheavy chain amino acid sequence of SEQ ID NO:2 or amino acid sequencevariants thereof which retain the ability to bind HER2 and inhibitgrowth of tumor cells which overexpress HER2 (FIGS. 3A and 3B; see alsoU.S. Pat. No. 5,677,171, expressly incorporated herein by reference).

“Anti-CD11a rhuMAb” or “CD11a” are used interchangeably herein to referto a humanized anti-CD11a antibody comprising the light chain amino acidsequence of SEQ ID NO:3 and the heavy chain amino acid sequence of SEQID NO:4 or amino acid sequence variants thereof which retain the abilityto bind LFA-1 and to inhibit certain T-cell dependent immune functions(FIGS. 4A and 4B; see also U.S. Pat. No. 5,622,700; WO 98/23761; Steppeet al., Transplant Intl. 4:3-7 (1991); and Hourmant et al.Transplantation 58:377-380 (1994); which references are expresslyincorporated herein y reference). Anti-CD11a antibodies further include,e.g., MHM24 [Hildreth et al., Eur. J. Immunol., 13: 202-208 (1983)],R3.1 (IgG1) [R. Rothlein, Boehringer Ingelheim Pharmaceuticals, Inc.,Ridgefield, Conn.], 25-3 (or 25.3), an IgG1 available from Immunotech,France [Olive et al., in Feldmann, ed., Human T cell Clones. A newApproach to Immune Regulation, Clifton, N.J., Humana, 1986 p. 173], KBA(IgG2a) [Nishimura et al., Cell. Immunol., 107: 32 (1987); Nishimura etal., ibid., 94: 122 (1985)], M7/15 (IgG2b) [Springer et al., Immunol.Rev., 68: 171 (1982)], IOT16 [Vermot Desroches et al., Scand. J.Immunol., 33: 277-286 (1991)], SPVL7 [Vermot Desroches et al., supra],and M17 (IgG2a), available from ATCC, which are rat anti-murine CD11aantibodies. Preferred anti-CD11a antibodies are the humanized antibodiesdescribed in U.S. Pat. No. 6,037,454. It is also generally preferredthat the anti-CD11a antibodies are not T-cell depleting antibodies, thatis, that the administration of the anti-CD11a antibody does not reducethe level of circulating T-cells.”

The “composition” to be purified herein comprises the polypeptide ofinterest and one or more impurities. The composition may be “partiallypurified” (i.e. having been subjected to one or more purification steps,such as by non-affinity chromatography described herein or may beobtained directly from a host cell or organism producing the polypeptide(e.g. the composition may comprise harvested cell culture fluid).

As used herein, “polypeptide” refers generally to peptides and proteinshaving more than about ten amino acids. Preferably, the polypeptide is amammalian protein, examples of which include renin; a growth hormone,including human growth hormone and bovine growth hormone; growth hormonereleasing factor; parathyroid hormone; thyroid stimulating hormone;lipoproteins; alpha-1-antitrypsin; insulin A-chain; insulin B-chain;proinsulin; follicle stimulating hormone; calcitonin; luteinizinghormone; glucagon; clotting factors such as factor VIIIC, factor IX,tissue factor, and von Willebrands factor; anti-clotting factors such asProtein C; atrial natriuretic factor; lung surfactant; a plasminogenactivator, such as urokinase or human urine or tissue-type plasminogenactivator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumornecrosis factor-alpha and -beta; enkephalinase; RANTES (regulated onactivation normally T-cell expressed and secreted); human macrophageinflammatory protein (MIP-1-alpha); a serum albumin such as human serumalbumin; Muellerian-inhibiting substance; relaxin A-chain; relaxinB-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbialprotein, such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyteassociated antigen (CTLA), such as CTLA-4; inhibin; activin; vascularendothelial growth factor (VEGF); receptors for hormones or growthfactors; Protein A or D; rheumatoid factors; a neurotrophic factor suchas bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or-6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-β;platelet-derived growth factor (PDGF); fibroblast growth factor such asaFGF and bFGF; epidermal growth factor (EGF); transforming growth factor(TGF) such as TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3,TGF-β4, or TGF-β5; insulin-like growth factor-I and -II (IGF-I andIGF-II); des(1-3)-IGF-I (brain IGF-1), insulin-like growth factorbinding proteins (IGFBPs); CD proteins such as CD3, CD4, CD8, CD19 CD20,CD34, and CD40; erythropoietin; osteoinductive factors; immunotoxins; abone morphogenetic protein (BMP); an interferon such asinterferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs),e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10;superoxide dismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments and/or variants of any of the above-listedpolypeptides. In addition, a protein or polypeptide of the invention isan antibody, fragment or variant thereof, that binds specifically to anyof the above-listed polypeptides.

An “impurity” is a material that is different from the desiredpolypeptide product or protein of interest. The impurity includes, butis not limited to, a host cell protein (HCP, such as CHOP), apolypeptide other than the target polypeptide, nucleic acid, endotoxinetc.

An “acidic variant” is a variant of a polypeptide of interest which ismore acidic (e.g. as determined by cation exchange chromatography) thanthe polypeptide of interest. An example of an acidic variant is adeamidated variant.

The term “protein of interest” and “target protein” are usedinterchangeably and refer to a protein or polypeptide such as anantibody (as defined herein) that is to be purified by a method of theinvention from a mixture of proteins and, optionally, other materialssuch as cell debris and the like.

The terms “Chinese hamster ovary cell protein” and “CHOP” are usedinterchangeably to refer to a mixture of host cell proteins (“HCP”)derived from a Chinese hamster ovary (“CHO”) cell culture. The HCP orCHOP is generally present as an impurity in a cell culture medium orlysate (e.g., a harvested cell culture fluid (“HCCF”)) comprising aprotein of interest such as an antibody or immunoadhesin expressed in aCHO cell.) The amount of CHOP present in a mixture comprising a proteinof interest provides a measure of the degree of purity for the proteinof interest. HCP or CHOP includes, but is not limited to, a protein ofinterest expressed by the host cell, such as a CHO host cell. Typically,the amount of CHOP in a protein mixture is expressed in parts permillion relative to the amount of the protein of interest in themixture. It is understood that where the host cell is another mammaliancell type, an E. coli, a yeast, an insect cell, or a plant cell, HCPrefers to the proteins, other than target protein, found in a lysate ofthe host cell.

The term “parts per million” or “ppm” are used interchangeably herein torefer to a measure of purity of the protein of interest purified by amethod of the invention. The units ppm refer to the amount of HCP orCHOP in nanograms/milliliter per protein of interest inmilligrams/milliliter (i.e., CHOP ppm ═(CHOP ng/ml)/(protein of interestmg/ml), where the proteins are in solution). Where the proteins aredried (such as by lyophilization), ppm refers to (CHOP ng)/(protein ofinterest mg)).

By “purifying” a polypeptide from a composition comprising thepolypeptide and one or more impurities is meant increasing the degree ofpurity of the polypeptide in the composition by removing (completely orpartially) at least one impurity from the composition. According to thepresent invention, purification is performed without the use of anaffinity chromatography step. A “purification step” may be part of anoverall purification process resulting in a “homogeneous” composition,which is used herein to refer to a composition comprising less than 100ppm HCP in a composition comprising the protein of interest,alternatively less than 90 ppm, less than 80 ppm, less than 70 ppm, lessthan 60 ppm, less than 50 ppm, less than 40 ppm, less than 30 ppm, lessthan 20 ppm, less than 10 ppm, less than 5 ppm, or less than 3 ppm.

The terms “Protein A” and “ProA” are used interchangeably herein andencompasses Protein A recovered from a native source thereof, Protein Aproduced synthetically (e.g. by peptide synthesis or by recombinanttechniques), and variants thereof which retain the ability to bindproteins which have a C_(H)2/C_(H)3 region, such as an Fc region.Protein A can be purchased commercially from Repligen, Pharmacia andFermatech. Protein A is generally immobilized on a solid phase supportmaterial. The term “ProA” also refers to an affinity chromatographyresin or column containing chromatographic solid support matrix to whichis covalently attached Protein A.

The term “chromatography” refers to the process by which a solute ofinterest in a mixture is separated from other solutes in a mixture as aresult of differences in rates at which the individual solutes of themixture migrate through a stationary medium under the influence of amoving phase, or in bind and elute processes.

The term “affinity chromatography” and “protein affinity chromatography”are used interchangeably herein and refer to a protein separationtechnique in which a protein of interest or antibody of interest isreversibly and specifically bound to a biospecific ligand. Preferably,the biospecific ligand is covalently attached to a chromatographic solidphase material and is accessible to the protein of interest in solutionas the solution contacts the chromatographic solid phase material. Theprotein of interest (e.g., antibody, enzyme, or receptor protein)retains its specific binding affinity for the biospecific ligand(antigen, substrate, cofactor, or hormone, for example) during thechromatographic steps, while other solutes and/or proteins in themixture do not bind appreciably or specifically to the ligand. Bindingof the protein of interest to the immobilized ligand allowscontaminating proteins or protein impurities to be passed through thechromatographic medium while the protein of interest remainsspecifically bound to the immobilized ligand on the solid phasematerial. The specifically bound protein of interest is then removed inactive form from the immobilized ligand with low pH, high pH, high salt,competing ligand, and the like, and passed through the chromatographiccolumn with the elution buffer, free of the contaminating proteins orprotein impurities that were earlier allowed to pass through the column.Any component can be used as a ligand for purifying its respectivespecific binding protein, e.g. antibody.

The terms “non-affinity chromatography” and “non-affinity purification”refer to a purification process in which affinity chromatography is notutilized. Non-affinity chromatography includes chromatographictechniques that rely on non-specific interactions between a molecule ofinterest (such as a protein, e.g. antibody) and a solid phase matrix.

The term “specific binding” as used herein, such as to describeinteractions between a molecule of interest and a ligand bound to asolid phase matrix, refers to the generally reversible binding of aprotein of interest to a ligand through the combined effects of spatialcomplementarity of protein and ligand structures at a binding sitecoupled with electrostatic forces, hydrogen bonding, hydrophobic forces,and/or van der Waals forces at the binding site. The greater the spatialcomplementarity and the stronger the other forces at the binding site,the greater will be the binding specificity of a protein for itsrespective ligand. Non-limiting examples of specific binding includesantibody-antigen binding, enzyme-substrate binding, enzyme-cofactorbinding, metal ion chelation, DNA binding protein-DNA binding,regulatory protein-protein interactions, and the like. Ideally, inaffinity chromatography specific binding occurs with an affinity ofabout 10⁻⁴ to 10⁻⁸ M in free solution.

The term “non-specific binding” as used herein, such as to describeinteractions between a molecule of interest and a ligand or othercompound bound to a solid phase matrix, refers to binding of a proteinof interest to the ligand or compound on a solid phase matrix throughelectrostatic forces, hydrogen bonding, hydrophobic forces, and/or vander Waals forces at an interaction site, but lacking structuralcomplementarity that enhances the effects of the non-structural forces.Examples of non-specific interactions include, but are not limited to,electrostatic, hydrophobic, and van der Waals forces as well as hydrogenbonding.

A “salt” is a compound formed by the interaction of an acid and a base.A salt useful for the invention include, but are not limited to acetate(e.g. sodium acetate), citrate (e.g. sodium citrate), chloride (e.g.sodium chloride), sulphate (e.g. sodium sulphate), or a potassium salt.

As used herein, “solvent” refers to a liquid substance capable ofdissolving or dispersing one or more other substances to provide asolution. Solvents include aqueous and organic solvents, where usefulorganic solvents include a non-polar solvent, ethanol, methanol,isopropanol, acetonitrile, hexylene glycol, propylene glycol, and2,2-thiodiglycol.

The term “detergent” refers to ionic and nonionic surfactants such aspolysorbates (e.g. polysorbates 20 or 80); poloxamers (e.g. poloxamer188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate;sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.), Useful detergents is a polysorbate, such as polysorbate20 (TWEEN 20®) or polysorbate 80 (TWEEN 80®).

A “polymer” herein is a molecule formed by covalent linkage of two ormore monomers, where the monomers are not amino acid residues. Examplesof polymers include polyethyl glycol, polypropyl glycol, and copolymers(e.g. Pluronics, PF68 etc). A useful polymer is polyethylene glycol(PEG), e.g. PEG 400 and PEG 8000.

The term “ion-exchange” and “ion-exchange chromatography” refers to thechromatographic process in which a solute of interest (such as aprotein) in a mixture interacts with a charged compound linked (such asby covalent attachment) to a solid phase ion exchange material such thatthe solute of interest interacts non-specifically with the chargedcompound more or less than solute impurities or contaminants in themixture. The contaminating solutes in the mixture elute from a column ofthe ion exchange material faster or slower than the solute of interestor are bound to or excluded from the resin relative to the solute ofinterest. “Ion-exchange chromatography” specifically includes cationexchange, anion exchange, and mixed mode chromatography.

The phrase “ion exchange material” refers to a solid phase that isnegatively charged (i.e. a cation exchange resin) or positively charged(i.e. an anion exchange resin). The charge may be provided by attachingone or more charged ligands to the solid phase, e.g. by covalentlinking. Alternatively, or in addition, the charge may be an inherentproperty of the solid phase (e.g. as is the case for silica, which hasan overall negative charge).

By “solid phase” is meant a non-aqueous matrix to which one or morecharged ligands can adhere. The solid phase may be a purificationcolumn, a discontinuous phase of discrete particles, a membrane, orfilter etc. Examples of materials for forming the solid phase includepolysaccharides (such as agarose and cellulose); and other mechanicallystable matrices such as silica (e.g. controlled pore glass),poly(styrenedivinyl)benzene, polyacrylamide, ceramic particles andderivatives of any of the above.

A “cation exchange resin” refers to a solid phase which is negativelycharged, and which thus has free cations for exchange with cations in anaqueous solution passed over or through the solid phase. A negativelycharged ligand attached to the solid phase to form the cation exchangeresin may, e.g., be a carboxylate or sulfonate. Commercially availablecation exchange resins include carboxy-methyl-cellulose, sulphopropyl(SP) immobilized on agarose (e.g. SP-SEPHAROSE FAST FLOW™ orSP-SEPHAROSE HIGH PERFORMANCE™, from Pharmacia) and sulphonylimmobilized on agarose (e.g. S-SEPHAROSE FAST FLOW™ from Pharmacia). A“mixed mode ion exchange resin” refers to a solid phase which iscovalently modified with cationic, anionic, and hydrophobic moieties. Acommercially available mixed mode ion exchange resin is BAKERBOND ABX™(J.T. Baker, Phillipsburg, N.J.) containing weak cation exchange groups,a low concentration of anion exchange groups, and hydrophobic ligandsattached to a silica gel solid phase support matrix.

The term “anion exchange resin” is used herein to refer to a solid phasewhich is positively charged, e.g. having one or more positively chargedligands, such as quaternary amino groups, attached thereto. Commerciallyavailable anion exchange resins include DEAE cellulose, QAE SEPHADEX™and FAST Q SEPHAROSE™ (Pharmacia).

A “buffer” is a solution that resists changes in pH by the action of itsacid-base conjugate components. Various buffers which can be employeddepending, for example, on the desired pH of the buffer are described inBuffers. A Guide for the Preparation and Use of Buffers in BiologicalSystems, Gueffroy, D., ed. Calbiochem Corporation (1975). In oneembodiment, the buffer has a pH in the range from about 2 to about 9,alternatively from about 3 to about 8, alternatively from about 4 toabout 7 alternatively from about 5 to about 7. Non-limiting examples ofbuffers that will control the pH in this range include MES, MOPS, MOPSO,Tris, HEPES, phosphate, acetate, citrate, succinate, and ammoniumbuffers, as well as combinations of these.

The “loading buffer” is that which is used to load the compositioncomprising the polypeptide molecule of interest and one or moreimpurities onto the ion exchange resin. The loading buffer has aconductivity and/or pH such that the polypeptide molecule of interest(and generally one or more impurities) is/are bound to the ion exchangeresin or such that the protein of interest flows through the columnwhile the impurities bind to the resin.

The “intermediate buffer” is used to elute one or more impurities fromthe ion exchange resin, prior to eluting the polypeptide molecule ofinterest. The conductivity and/or pH of the intermediate buffer is/aresuch that one or more impurity is eluted from the ion exchange resin,but not significant amounts of the polypeptide of interest.

The term “wash buffer” when used herein refers to a buffer used to washor re-equilibrate the ion exchange resin, prior to eluting thepolypeptide molecule of interest. Conveniently, the wash buffer andloading buffer may be the same, but this is not required.

The “elution buffer” is used to elute the polypeptide of interest fromthe solid phase. The conductivity and/or pH of the elution buffer is/aresuch that the polypeptide of interest is eluted from the ion exchangeresin.

A “regeneration buffer” may be used to regenerate the ion exchange resinsuch that it can be re-used. The regeneration buffer has a conductivityand/or pH as required to remove substantially all impurities and thepolypeptide of interest from the ion exchange resin.

The term “conductivity” refers to the ability of an aqueous solution toconduct an electric current between two electrodes. In solution, thecurrent flows by ion transport. Therefore, with an increasing amount ofions present in the aqueous solution, the solution will have a higherconductivity. The unit of measurement for conductivity is milliSeimensper centimeter (mS/cm), and can be measured using a conductivity metersold, e.g., by Orion. The conductivity of a solution may be altered bychanging the concentration of ions therein. For example, theconcentration of a buffering agent and/or concentration of a salt (e.g.NaCl or KCl) in the solution may be altered in order to achieve thedesired conductivity. Preferably, the salt concentration of the variousbuffers is modified to achieve the desired conductivity as in theExample below.

The “pI” or “isoelectric point” of a polypeptide refer to the pH atwhich the polypeptide's positive charge balances its negative charge. pIcan be calculated from the net charge of the amino acid residues orsialic acid residues of attached carbohydrates of the polypeptide or canbe determined by isoelectric focusing.

By “binding” a molecule to an ion exchange material is meant exposingthe molecule to the ion exchange material under appropriate conditions(pH/conductivity) such that the molecule is reversibly immobilized in oron the ion exchange material by virtue of ionic interactions between themolecule and a charged group or charged groups of the ion exchangematerial.

By “washing” the ion exchange material is meant passing an appropriatebuffer through or over the ion exchange material.

To “elute” a molecule (e.g. polypeptide or impurity) from an ionexchange material is meant to remove the molecule therefrom by alteringthe ionic strength of the buffer surrounding the ion exchange materialsuch that the buffer competes with the molecule for the charged sites onthe ion exchange material.

As used herein, “filtrate” refers to that portion of a sample thatpasses through the filtration membrane.

As used herein, “retentate” refers to that portion of a sample that issubstantially retained by the filtration membrane.

Tangential flow filtration” or “TFF” or “crossflow filtration” refers toa filtration process in which the sample mixture circulates across thetop of the membrane, while applied pressure causes certain solutes andsmall molecules to pass through the membrane. Typically, the solutionflows parallel to the filter membrane. A pressure differential acrossthe membrane causes fluid and filterable solutes to flow through thefilter. This can be conducted as a continuous-flow process, since thesolution is passed repeatedly over the membrane while that fluid thatpasses through the filter is continually drawn off into a separatecircuit.

“High performance tangential flow filtration” or “HPTFF” refers to TFFperformed at a flux between 5% and 100% of the transmembrane pressure onthe flux versus transmembrane pressure curve (see, for example, vanReis, R. U.S. Pat. No. 5,256,694; U.S. Pat. No. 4,490,937; and U.S. Pat.No. 6,054,051).

As used herein, “lysate impurities” refers to all undesired componentsof a mixture in which the desired plasmid DNA is contained, includingchromosomal DNA, host proteins, cell debris, secreted host cellproteins, including cell membrane debris, carbohydrates, small degradednucleotides, host RNA, lipopolysaccharides, etc.

“Cellulose membrane” refers to a cellulose polymer, where the celluloseis repeating units of D-glucose. The primary alcohol group of a glucosemonomer provides the reactive species on the membrane to which thecharged compound is covalently attached.

“CRC membrane” refers to a composite regenerated cellulose membraneprepared by casting cellulose on a microporous substrate to control theaverage pore size and limit the number of defects in the cellulosesheet.

“Charged compound” refers to the compound linked to the filtrationmembrane, wherein the compound comprises a moiety having a positive ornegative charge under the conditions used to separate a protein from amixture of proteins. According to the invention, the charged compoundmay further comprise a linker arm between the membrane and the chargedmoiety such that the charged compound projects from the surface of themembrane. Where the charged compound projects from the surface of a poreinto the lumen of the pore, the charged compound modifies the effectivesize of the pore and modifies the pore size distribution of themembrane.

“Reactive charged compound” refers to the charged compound prior tolinkage to the membrane, such that the reactive charged compound stillretains the reactive moiety that promotes the membrane-reactive chargedcompound reaction. For example, where the charged compound is a propyltrimethyl ammonium ion covalently attached to a cellulose membrane, thereactive charged compound may be bromopropyl trimethyl ammonium bromide.The covalent attachment involves nucleophilic displacement of the alkylbromine by a primary alcohol of the cellulose matrix.

“Linker arm” refers to the portion of the charged compound moleculebetween the portion that reacts or has reacted with a reactive group onthe surface of a filtration membrane and the charged moiety. Preferably,the linker arm is a chain of atoms or molecular subunits, which chain isinert to the reaction conditions used to covalently link the chargedcompound to the membrane, and is further inert to the aqueous conditionsused during protein separation. A linker arm may comprise, but is notlimited to, an alkyl chain of from one to twenty carbon atoms, acarbohydrate chain of from one to fifteen saccharide moities (including,for example, ribose and deoxyribose), a dextran chain of from one tofifteen saccharide moities, an amino acid chain of from one to twentyfive amino acids, and other polymers (such as those used to manufacturethe membrane itself) of from one to twenty five repeat units. Where acharged compound comprises an amino acid chain as a linker arm and thecharged moiety is the terminal amino acid of the chain, the side chainof the terminal amino acid is preferably a charged side chain.

“Sieving” refers to the ratio of the concentration of a particularsolute in the filtrate (downstream of the membrane) to the concentrationof the same solute in the feed solution (upsteam of the membrane) (seeZeman and Zydney, supra, p. 308). Generally a high sieving valuesuggests that the solute readily passes through the membrane, while alow sieving value suggests that the solute is largely retained by themembrane. Where it is desired to retain a solute upstream of themembrane, a reduced sieving coefficient is preferred.

“Permeability” refers to the filtration rate divided by the net pressuredrop across the membrane. Permeability is therefore the inverse ofmembrane resistance. Membrane permeability is primarily determined bypore size distribution, porosity (pore density), membrane thickness, andsolvent viscosity. Generally, as permeability increases, sievingincreases. When sieving is improved due to the addition of a chargedcompound to the membrane, the sieving improvement is an improvementrelative to a membrane having substantially the same permeability as thecharged membrane, but lacking the charged compound. Thus, where theimprovement is a reduction in sieving because a charged solute, such asa protein, is retained by a like-charged membrane, the sieving is areduction at comparable or substantially the same permeability.Consequently, the rate of filtration is maintained, while theselectivity of the membrane is improved.

“Pore size distribution” refers, basically, to the number of poreshaving an actual radius, R, near some theoretical radius, r, expressedas the probability density function (see, Zeman, L. J. and Zydney, A.L., supra, p. 299-301). As the standard deviation of actual pore radiiincreases, the pore size distribution increases. Narrowed pore sizedistribution results from a reduction in the standard deviation of thepores from the theoretical value. This is achieved, for example, whenthe sizes of some of the larger pores are reduced by addition of chargedcompound into the larger pores of a charged membrane. The principle ofliquid-liquid pore intrusion is useful for measuring pore sizedistribution (see R. van Reis and A. L. Zydney, supra, p. 2201).According to this principle, two highly immiscible liquids, such assolutions of a sulfate salt and a poly(ethylene glycol) are contactedthrough mixing to reach equilibrium partitioning. The membrane to betested is primed with one of the liquids so that all pores are filled.After draining the feed channels, the second fluid is introduced intothe system. The first fluid is then displaced out of the pores by thesecond fluid, and the flow rate is measured as a function oftrans-membrane pressure. The resulting data provide information on poresize distribution and can be correlated with the nominal molecularweight cutoff (see R. van Reis and A. L. Zydney, supra, p. 2201).

“Net charge” when referring to a membrane or protein charge is meant acharge that is predominately positive or negative, but does not refer toa specific value for the number of positive charges versus the number ofnegative charges on the membrane or protein, unless otherwise noted.Similarly, “like charge” and “same charge” refer to the situation inwhich a protein having a given charge, positive or negative, is comparedto a membrane or other protein having a given charge, either positive ornegative.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

A “disorder” is any condition that would benefit from treatment with thepolypeptide purified as described herein. This includes chronic andacute disorders or diseases including those pathological conditionswhich predispose the mammal to the disorder in question.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to thepolypeptide. The label may itself be detectable (e.g., radioisotopelabels or fluorescent labels) or, in the case of an enzymatic label, maycatalyze chemical alteration of a substrate compound or compositionwhich is detectable.

B. Modes for Carrying Out the Invention

1. Protein Purification

Manufacturers of protein-based pharmaceutical products must comply withstrict regulatory standards, including extremely stringent purityrequirements. To ensure safety, regulatory agencies, such as the Foodand Drug Administration (FDA), require that protein-basedpharmaceuticals, including those produced by recombinant DNA technology,be substantially free from impurities, such as host cell proteins,viruses, DNA, endotoxins, aggregates, fragments, and variants of therecombinant protein, and the like. While various protein purificationprotocols are available and widely used in the pharmaceutical industry,they typically include affinity-purification, such as Protein Apurification in the case of antibodies, in order to reach the requireddegree of purity. As indicated herein above, although Protein A affinityremoves more than 99.5% of impurities, this benefit comes at a price.Protein A is significantly more expensive than the price of non-affinitymedia, and Protein A-based purification methods often raise issuesassociated with resin stability, cleanability and lifetime, ligandleakage, and the potential immunogenicity of Protein A residuescontaminating the purified product.

The present invention involves the purification of proteins, inparticular recombinant proteins, by a protocol not including an affinitychromatography step. More specifically, the invention provides methodsfor the purification of (recombinant) proteins, including but notlimited to antibodies, by steps not including affinity chromatography,to a degree that allows direct use of the purified proteins in humantherapy, thereby eliminating costly affinity chromatography steps aswell as a final ultrafiltration diafiltration frequently required toconcentration and formulate a therapeutic protein.

The present invention is based on experimental findings demonstratingthat recombinant proteins can be purified from a mixture comprising hostcell proteins by purification schemes not employing affinitychromatography to the same degree as processes incorporating an affinitychromatography step. In particular, it was found that a three-stepnon-affinity purification process, including two non-affinitychromatography steps followed by high performance tangential flowfiltration (HPTFF) as the last step, can yield a high-purity productthat contains host cell protein impurities in an amount less than 100parts per million (ppm).

The protein to be purified using the method described herein isgenerally produced using recombinant techniques. Methods for producingrecombinant proteins are described, e.g., in U.S. Pat. Nos. 5,534,615and 4,816,567, specifically incorporated herein by reference. Inpreferred embodiments, the protein of interest is produced in a CHO cell(see, e.g. WO 94/11026). Examples of proteins which can be purifiedusing the process described herein have been described above.

When using recombinant techniques, the protein can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the protein is produced intracellularly, as a first step, theparticulate debris, either host cells or lysed fragments, is removed,for example, by centrifugation or filtration. Where the protein issecreted into the medium, the recombinant host cells may be separatedfrom the cell culture medium by tangential flow filtration, for example.

Once a mixture containing the protein of interest has been obtained, itsseparation from the other proteins produced by the cell is usuallyperformed using a combination of different chromatography techniques.These techniques separate mixtures of proteins on the basis of theircharge, degree of hydrophobicity, or size. Several differentchromatography resins are available for each of these techniques,allowing accurate tailoring of the purification scheme to the particularprotein involved. The essence of each of these separation methods isthat proteins can be caused either to move at different rates down along column, achieving a physical separation that increases as they passfurther down the column, or to adhere selectively to the separationmedium, being then differentially eluted by different buffers. In somecases, the desired protein is separated from impurities when theimpurities specifically adhere to the column, and the protein ofinterest does not, that is, the protein of interest is present in the“flow-through”.

As noted before, according to the present invention, proteins can bepurified to a degree characterized by the presence of less than 100 ppmhost cell protein impuritites by one or two non-affinity purificationsteps, followed by HPTFF. The non-affinity purification steps may bebased on non-affinity chromatography, or may include non-chromatographicpurification techniques.

Exemplary non-affinity chromatography purification steps includehydroxyapatite chromatography; hydrophobic interaction chromatography(HIC); reverse phase HPLC; chromatography on silica; chromatofocusing;and gel filtration; cation exchange (e.g., SP-Sepharose) chromatography;anion exchange (e.g., Q-Sepharose) chromatography, mixed modechromatography (e.g., ABx), and hydrophobic charge inductionchromatography.

Exemplary non-affinity, non-chromatographic purification steps includedialysis; ammonium sulphate precipitation; and ethanol precipitation.

In a preferred embodiment, the process of the present invention includestwo chromatographic non-affinity separation steps, followed by HPTTF,optionally charged membrane HPTFF. In another preferred embodiment, thechromatographic non-affinity separation steps are selected from cationexchange chromatography, anion exchange chromatography, mixed mode ionexchange chromatography, hydrophobic interaction chromatography (HIC),and hydrophobic charge induction chromatography (HCI). In anotherpreferred embodiment, the purification protocol includes the steps of(1) cation exchange chromatography, (2) anion exchange chromatography,and (3) HPTFF in this order, in the absence of any affinity purificationsteps, and, preferably, without further purification steps of any kind.

Ion exchange chromatography is a chromatographic technique that iscommonly used for the purification of proteins. In ion exchangechromatography, charged patches on the surface of the solute areattracted by opposite charges attached to a chromatography matrix,provided the ionic strength of the surrounding buffer is low. Elution isgenerally achieved by increasing the ionic strength (i.e. conductivity)of the buffer to compete with the solute for the charged sites of theion exchange matrix. Changing the pH and thereby altering the charge ofthe solute is another way to achieve elution of the solute. The changein conductivity or pH may be gradual (gradient elution) or stepwise(step elution). In the past, these changes have been progressive; i.e.,the pH or conductivity is increased or decreased in a single direction.

The impure preparation derived from the recombinant host cells is loadedon the equilibrated chromatography solid phase matrix using a loadingbuffer which may be the same as the equilibration buffer. As the impurepreparation flows through the solid phase, the protein and otherimpurities (such as Chinese Hamster Ovary Proteins, CHOP, where theprotein is produced in a CHO cell) bind differentially to the solidphase thereby effecting separation as the proteins pass through thechromatography column.

The amount and type of buffer, salt, and/or other compound in the buffercomposition are such that the combined amount elutes the proteinimpurity(ies) differentially from the protein of interest, where theprotein of interest may be retained relative to the impurities or theimpurities are retained relative to the protein of interest. Bufferssalts and other additives useful in practicing the invention includewithout limitation buffer salts such as acetate, citrate, histidine,phosphate, ammonium acetate, MES, CHAPS, MOPSO, Tris, and the like;salts for adjusting buffer ionic strength such as sodium chloride andpotassium chloride; and other additives such as amino acids (such asglycine and histidine), chaotropes (such as urea), alcohols (such asethanol, mannitol, glycerol, and benzyl alcohol), detergent (such asTween™ and C12E8), and sugars (such as sucrose, mannitol, maltose,trehalose, glucose, and fructose). Any of these buffers and additivesand the concentrations used may vary according to the type ofchromatography practiced, which buffer and additive compositions andconcentrations are readily determined by standard methods.

The pH of the elution buffer may be from about 2 to about 9,alternatively from about 3 to about 8, from about 4 to about 8, or fromabout 5 to about 8, although the pH or pH range for elution will bedetermined according to the protein of interest and the type ofchromatography and HPTFF practiced. Appropriate pH ranges for a loading,wash, or elution buffer are readily determined by standard methods suchthat the protein of interest is recovered in an active form. Examples ofelution buffers for this purpose include citrate or acetate buffers.

The ionic strength of a buffer (measured as conductivity, for example)may be from about 0.2-20 mS/cm, alternatively from about 0.2-8 mS/cm,from about 0.2-6 mS/cm, from about 0.2-4 mS/cm, from about 0.2-2 mS/cm,or from about 1-2 mS/cm, although the ionic strength or ionic strengthrange for a load, wash, or elution buffer will be determined accordingto the protein of interest and the type of chromatography practiced anddiafiltration buffers for the HPTFF method practiced. Appropriate ionicstrength ranges for a buffer are readily determined by standard methodssuch that the protein of interest is recovered in an active form.

The cation exchange chromatography step typically removes at least partof the host cell proteins, e.g. CHOP, if the protein was produced in CHOcells, and variants, degradation products, and aggregates of the proteinto be purified. The anion exchange chromatography step further purifiesthe protein from the remaining host cell proteins, e.g. CHOP, variants,degradation products, and aggregates of the protein, and also fromendotoxins and DNA impurities.

Following non-affinity chromatography or other non-affinitypurification, the eluted protein of interest is subject to HPTFF. HPTFFis a two-dimensional unit operation that selectively separates soluteson the basis of both size and charge. HPTFF is able to provide the highselectivity required for effective protein purification by exploitingseveral recent developments. First, unlike traditional membraneprocesses, HPTFF is operated in the pressure-dependent regime underconditions to minimize fouling, exploit concentration polarization,optimize separation by maintaining a nearly uniform flux andtransmembrane pressure throughout the separation module (van Reis, R.,U.S. Pat. Nos. 5,256,694; 5,490,937; and 6,054,051, supra). Separationselectivity can be improved by controlling filtrate buffer pH and ionicstrength to maximize differences in effective volume of the differentspecies in a mixture (van Reis et al. (2001), supra; van Reis et al.(1997), supra; and Saksena, S. and Zydney, A. L., Biotechnol. Bioeng.43:960-968 (1994)). In addition, the electrical charge of the membranecan be modified to increase the electrostatic exclusion of all specieswith like charge. Thus, a positively charged membrane will reject apositively charged protein to a greater extent than a negatively chargedmembrane of a similar pore size (van Reis et al. (2001), supra; Nakao,S. et al., Desalination 70:191-205 (1988); and van Reis et al., J.Membr. Sci. 159:133-142 (1999)). Further, protein separations in HPTFFare accomplished using a diafiltration mode in which the impurity (orproduct) is washed out of the retentate by simultaneously adding freshbuffer to the feed reservoir as filtrate is removed through themembrane. This buffer addition maintains an appropriate proteinconcentration in the retentate throughout the separation. Diafiltrationalso makes it possible to obtain purification factors for productscollected in the retentate that are greater than the membraneselectivity due to the continual removal of impurities in the filtrate(van Reis et al. (2001), supra; and van Reis, R. and Saksena, S., J.Membr. Sci. 129:19-29 (1997)).

An HPTFF filtration membrane useful for protein separations is asynthetic (frequently polymeric) selective barrier for industrial orlab-scale ultrafiltration (UF) (see Leos J. Zeman and Andrew L. Zydney,“Microfiltration and Ultrafiltration: Principles and Applications,”1996, Marcel Dekker, Inc., p. 3). In these processes, certain feedstream components, such as proteins, pass through pores of the membraneinto a filtrate, while other, usually larger, proteins or components areretained by the membrane in the retentate (see Zeman and Zydney, supra,p. 3).

Protein ultrafiltration is a pressure-driven membrane process used forthe concentration or purification of protein solutions (Robert van Reisand Andrew L. Zydney, “Protein Ultrafiltration” in Encyclopedia ofBioprocess Technology: Fermentation, Biocatalysis, and Bioseparation, M.C. Flickinger and S. W. Drew, eds., John Wiley & Sons, Inc. (1999), p.2197). UF membranes typically have a mean pore size between 10 and 500Angstroms, which is between the mean pore size of reverse osmosis andmicrofiltration membranes. Ultrafiltration separates solutes based ondifferences in the rate of filtration of different components across themembrane in response to a given pressure driving force (R. van Reis andA. L. Zydney, supra, p. 2197). Solute filtration rates, and thusmembrane selectivity, are determined by both thermodynamic andhydrodynamic interactions (R. van Reis and A. L. Zydney, supra, p.2197). Ultrafiltration is frequently used in downstream processing forprotein concentration, buffer exchange and desalting, proteinpurification, virus clearance, and clarification (R. van Reis and A. L.Zydney, supra, p. 2197).

Using HPTFF, the desired protein is collected in either the retentate orfiltrate depending on the relative filtration rates (R. van Reis and A.L. Zydney, supra, p. 2197). HPTFF is useful for separating proteins ofsimilar size using the above-described semipermeable membranes (See, forexample, R. van Reis, et al., Biotech. Bioeng. 56:71-82 (1997) and R.van Reis et al., J. Memb. Sci. 159:133-142 (1999)). HPTFF achieves highselectivity by controlling filtrate flux and device fluid mechanics inorder to minimize fouling and exploit the effects of concentrationpolarization (R. van Reis et al., J. Memb. Sci. 159:133-142 (1999)).

The performance of HPTFF can be evaluated by two parameters, selectivityand throughput, which are used to optimize process yield andpurification factor (van Reis and Saksena, supra, 1997; van Reis et al.,supra, 1999). The selectivity is defined as the ratio of the observedsieving coefficients of the permeable and retained solutes. Since thepresent HPTFF application is based on the retention of a target proteinand the sieving of HCP, or impurities, the selectivity is described as:$\begin{matrix}{\Psi = \frac{S_{HCP}}{S_{Targetprotein}}} & {{Equation}\quad 1}\end{matrix}$

where the sieving coefficient is defined as the dimensionless ratio:$\begin{matrix}{S = \frac{C_{filtrate}}{C_{Feed}}} & {{Equation}\quad 2}\end{matrix}$

with C_(filtrate) and C_(feed) the solute concentrations in the filtrateand in the feed lines.

The throughput is defined as the product of the filtrate flux and thedifference in sieving between the permeable and retained solutes:J·ΔS=J·(S _(HCP) −S _(T arg etprotein))  Equation 3

Another calculated process parameter is the retentate yield duringconstant volume diafiltration. This yield is expressed as:Y=e^(−NS) ^(T arg etprotein)   Equation 4

where N equals the number of diavolumes and S equals the sieving of theconsidered solute.

Further details of the HPTFF purification steps will be provided in theExamples below.

The preferred measure of protein purification by the process of thepresent invention is the measure of host cell protein impurity, e.g.CHOP impurity, where the recombinant protein to be purified is producedin CHO cells. The purified protein preferably should contain less than100, more preferably less than 90, less than 80, less than 70, less than60, less than 50, less than 40, less than 30, less than 20, less than10, less than 5 ppm, or less than 3 ppm of host cell proteins, e.g.CHOP, where the ppm values are calculated as defined above.

The protein thus recovered may be formulated in a pharmaceuticallyacceptable carrier and is used for various diagnostic, therapeutic orother uses known for such molecules.

2. Antibodies

The preferred protein to be purified according to the present inventionis an antibody. In particular, as described in the Examples below, forpurification of recombinant humanized monoclonal antibody (RhuMAb),conditioned Harvested Cell Culture Fluid (HCCF) from chinese hamsterovary (CHO) cells expressing RhuMAb was loaded onto an initial cationexchange column (SP-Sepharose Fast Flow Resin, Amersham Biosciences;(S)). The material collected from the S column, the S pool, was 15′collected from the SP-Sepharose column, conditioned and then loaded ontoan anion exchange (Q-Sepharose Fast Flow resin, Amersham Biosciences;(O)). The material collected from the anion exchange column (such as theQ column, as each the Q pool) was further purified by HPTFF.

Antibodies within the scope of the present invention include, but arenot limited to: anti-HER2 antibodies including Trastuzumab (HERCEPTIN®)(Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285-4289 (1992), U.S.Pat. No. 5,725,856); anti-CD20 antibodies such as chimeric anti-CD20“C2B8” as in U.S. Pat. No. 5,736,137 (RITUXAN®), a chimeric or humanizedvariant of the 2H7 antibody as in U.S. Pat. No. 5,721,108, B1, orTositumomab (BEXXAR®); anti-IL-8 (St John et al., Chest, 103:932 (1993),and International Publication No. WO 95/23865); anti-VEGF antibodiesincluding humanized and/or affinity matured anti-VEGF antibodies such asthe humanized anti-VEGF antibody huA4.6.1 AVASTIN® (Kim et al., GrowthFactors, 7:53-64 (1992), International Publication No. WO 96/30046, andWO 98/45331, published Oct. 15, 1998); anti-PSCA antibodies(WO01/40309); anti-CD40 antibodies, including S2C6 and humanizedvariants thereof (WO00/75348); anti-CD11a (U.S. Pat. No. 5,622,700, WO98/23761, Steppe et al., Transplant Intl. 4:3-7 (1991), and Hourmant etal., Transplantation 58:377-380 (1994)); anti-IgE (Presta et al., JImmunol. 151:2623-2632 (1993), and International Publication No. WO95/19181); anti-CD18 (U.S. Pat. No. 5,622,700, issued Apr. 22, 1997, oras in WO 97/26912, published Jul. 31, 1997); anti-IgE (including E25,E26 and E27; U.S. Pat. No. 5,714,338, issued Feb. 3, 1998 or U.S. Pat.No. 5,091,313, issued Feb. 25, 1992, WO 93/04173 published Mar. 4, 1993,or International Application No. PCT/US98/13410 filed Jun. 30, 1998,U.S. Pat. No. 5,714,338); anti-Apo-2 receptor antibody (WO 98/51793published Nov. 19, 1998); anti-TNF-α antibodies including cA2(REMICADE®), CDP571 and MAK-195 (See, U.S. Pat. No. 5,672,347 issuedSep. 30, 1997, Lorenz et al. J. Immunol. 156(4):1646-1653 (1996), andDhainaut et al. Crit. Care Med. 23(9):1461-1469 (1995)); anti-TissueFactor (TF) (European Patent No. 0 420 937 B1 granted Nov. 9, 1994);anti-human α₄β₇ integrin (WO 98/06248 published Feb. 19, 1998);anti-EGFR (chimerized or humanized 225 antibody as in WO 96/40210published Dec. 19, 1996); anti-CD3 antibodies such as OKT3 (U.S. Pat.No. 4,515,893 issued May 7, 1985); anti-CD25 or anti-tac antibodies suchas CHI-621 (SIMULECT®) and (ZENAPAX®) (See U.S. Pat. No. 5,693,762issued Dec. 2, 1997); anti-CD4 antibodies such as the cM-7412 antibody(Choy et al. Arthritis Rheum 39(1):52-56 (1996)); anti-CD52 antibodiessuch as CAMPATH-1H (Riechmann et al. Nature 332:323-337 (1988)); anti-Fcreceptor antibodies such as the M22 antibody directed against FcγRI asin Graziano et al. J. Immunol. 155(10):4996-5002 (1995);anti-carcinoembryonic antigen (CEA) antibodies such as hMN-14 (Sharkeyet al. Cancer Res. 55(23Suppl): 5935s-5945s (1995); antibodies directedagainst breast epithelial cells including huBrE-3, hu-Mc 3 and CHL6(Ceriani et al. Cancer Res. 55(23): 5852s-5856s (1995); and Richman etal. Cancer Res. 55(23 Supp): 5916s-5920s (1995)); antibodies that bindto colon carcinoma cells such as C242 (Litton et al. Eur J. Immunol.26(1):1-9 (1996)); anti-CD38 antibodies, e.g. AT 13/5 (Ellis et al. JImmunol. 155(2):925-937 (1995)); anti-CD33 antibodies such as Hu M195(Jurcic et al. Cancer Res 55(23 Suppl):5908s-5910s (1995) and CMA-676 orCDP771; anti-CD22 antibodies such as LL2 or LymphoCide (Juweid et al.Cancer Res 55(23 Suppl):5899s-5907s (1995)); anti-EpCAM antibodies suchas 17-1A (PANOREX®); anti-GpIIb/IIIa antibodies such as abciximab orc7E3 Fab (REOPRO®); anti-RSV antibodies such as MEDI-493 (SYNAGIS®);anti-CMV antibodies such as PROTOVIR®; anti-HIV antibodies such asPRO542; anti-hepatitis antibodies such as the anti-Hep B antibodyOSTAVIR®; anti-CA 125 antibody OvaRex; anti-idiotypic GD3 epitopeantibody BEC2; anti-αvβ3 antibody VITAXIN®; anti-human renal cellcarcinoma antibody such as ch-G250; ING-1; anti-human 17-1A antibody(3622W94); anti-human colorectal tumor antibody (A33); anti-humanmelanoma antibody R24 directed against GD3 ganglioside; anti-humansquamous-cell carcinoma (SF-25); and anti-human leukocyte antigen (HLA)antibodies such as Smart ID10 and the anti-HLA DR antibody Oncolym(Lym-1). The preferred target antigens for the antibody herein are: HER2receptor, VEGF, IgE, CD20, CD11a, and CD40.

Aside from the antibodies specifically identified above, the skilledpractitioner could generate antibodies directed against an antigen ofinterest, e.g., using the techniques described below.

(a) Antigen Selection and Preparation

The antibody herein is directed against an antigen of interest.Preferably, the antigen is a biologically important polypeptide andadministration of the antibody to a mammal suffering from a disease ordisorder can result in a therapeutic benefit in that mammal. However,antibodies directed against nonpolypeptide antigens (such astumor-associated glycolipid antigens; see U.S. Pat. No. 5,091,178) arealso contemplated. Where the antigen is a polypeptide, it may be atransmembrane molecule (e.g. receptor) or ligand such as a growthfactor. Exemplary antigens include those proteins described in section(3) below. Exemplary molecular targets for antibodies encompassed by thepresent invention include CD proteins such as CD3, CD4, CD8, CD19, CD20,CD22, CD34, CD40; members of the ErbB receptor family such as the EGFreceptor, HER2, HER3 or HER4 receptor; cell adhesion molecules such asLFA-1, Mac1, p150,95, VLA-4, ICAM-1, VCAM and αv/β3 integrin includingeither α or β subunits thereof (e.g. anti-CD11a, anti-CD18 or anti-CD11bantibodies); growth factors such as VEGF; IgE; blood group antigens;flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; proteinC, or any of the other antigens mentioned herein. Antigens to which theantibodies listed above bind are specifically included within the scopeherein.

Soluble antigens or fragments thereof, optionally conjugated to othermolecules, can be used as immunogens for generating antibodies. Fortransmembrane molecules, such as receptors, fragments of these (e.g. theextracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.cancer cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule.

Other antigens and forms thereof useful for preparing antibodies will beapparent to those in the art.

(b) Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the antigen to aprotein that is immunogenic in the species to be immunized, e.g.,keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of antigen or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

(c) Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, Protein A-Sepharose, hydroxyapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. Preferably theProtein A chromatography procedure described herein is used.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

In a further embodiment, monoclonal antibodies can be isolated fromantibody phage libraries generated using the techniques described inMcCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991)describe the isolation of murine and human antibodies, respectively,using phage libraries. Subsequent publications describe the productionof high affinity (nM range) human antibodies by chain shuffling (Markset al., Bio/Technology, 10:779-783 (1992)), as well as combinatorialinfection and in vivo recombination as a strategy for constructing verylarge phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266(1993)). Thus, these techniques are viable alternatives to traditionalhybridoma techniques for isolation of monoclonal antibodies.

(d) Humanized and Human Antibodies

A humanized antibody has one or more amino acid residues introduced intoit from a source which is non-human. These non-human amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman FR for the humanized antibody (Sims et al., J. Immunol., 151:2296(1993)). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993); and Duchosal et al. Nature 355:258(1992). Human antibodies can also be derived from phage-displaylibraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks etal., J. Mol. Biol., 222:581-597 (1991); Vaughan et al. Nature Biotech14:309 (1996)).

(e) Antibody Fragments

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992) and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See WO93/16185.

(e) Multispecific Antibodies

Multispecific antibodies have binding specificities for at least twodifferent antigens. While such molecules normally will only bind twoantigens (i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by thisexpression when used herein.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to another approach described in WO96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the C_(H)3domain of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).Alternatively, the antibodies can be “linear antibodies” as described inZapata et al. Protein Eng. 8(10):1057-1062 (1995). Briefly, theseantibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

3. Immunoadhesins

The simplest and most straightforward immunoadhesin design combines thebinding domain(s) of the adhesin (e.g. the extracellular domain (ECD) ofa receptor) with the hinge and Fc regions of an immunoglobulin heavychain. Ordinarily, when preparing the immunoadhesins of the presentinvention, nucleic acid encoding the binding domain of the adhesin willbe fused C-terminally to nucleic acid encoding the N-terminus of animmunoglobulin constant domain sequence, however N-terminal fusions arealso possible.

Typically, in such fusions the encoded chimeric polypeptide will retainat least functionally active hinge, C_(H)2 and C_(H)3 domains of theconstant region of an immunoglobulin heavy chain. Fusions are also madeto the C-terminus of the Fc portion of a constant domain, or immediatelyN-terminal to the C_(H)1 of the heavy chain or the corresponding regionof the light chain. The precise site at which the fusion is made is notcritical; particular sites are well known and may be selected in orderto optimize the biological activity, secretion, or bindingcharacteristics of the immunoadhesin.

In a preferred embodiment, the adhesin sequence is fused to theN-terminus of the Fc domain of immunoglobulin G₁ (IgG₁). It is possibleto fuse the entire heavy chain constant region to the adhesin sequence.However, more preferably, a sequence beginning in the hinge region justupstream of the papain cleavage site which defines IgG Fc chemically(i.e. residue 216, taking the first residue of heavy chain constantregion to be 114), or analogous sites of other immunoglobulins is usedin the fusion. In a particularly preferred embodiment, the adhesin aminoacid sequence is fused to (a) the hinge region and C_(H) ² and C_(H)3 or(b) the C_(H)1, hinge, C_(H)2 and C_(H)3 domains, of an IgG heavy chain.

For bispecific immunoadhesins, the immunoadhesins are assembled asmultimers, and particularly as heterodimers or heterotetramers.Generally, these assembled immunoglobulins will have known unitstructures. A basic four chain structural unit is the form in which IgG,IgD, and IgE exist. A four chain unit is repeated in the highermolecular weight immunoglobulins; IgM generally exists as a pentamer offour basic units held together by disulfide bonds. IgA globulin, andoccasionally IgG globulin, may also exist in multimeric form in serum.In the case of multimer, each of the four units may be the same ordifferent.

Various exemplary assembled immunoadhesins within the scope herein areschematically diagrammed below:

(a) AC_(L)-AC_(L);

(b) AC_(H)-(AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H), orV_(L)C_(L)-AC_(H));

(c) AC_(L)-AC_(H)-(AC_(L)-AC_(H), AC_(L)-V_(H)C_(H), V_(L)C_(L)-AC_(H),or V_(L)C_(L)-V_(H)C_(H))

(d) AC_(L)-V_(H)C_(H)-(AC_(H), or AC_(L)-V_(H)C_(H), orV_(L)C_(L)-AC_(H));

(e) V_(L)C_(L)-AC_(H)-(AC_(L)-V_(H)C_(H), or V_(L)C_(L)-AC_(H)); and

(f) (A-Y)_(n)-(V_(L)C_(L)-V_(H)C_(H))₂,

wherein each A represents identical or different adhesin amino acidsequences;

V_(L) is an immunoglobulin light chain variable domain;

V_(H) is an immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H) is an immunoglobulin heavy chain constant domain;

n is an integer greater than 1;

Y designates the residue of a covalent cross-linking agent.

In the interests of brevity, the foregoing structures only show keyfeatures; they do not indicate joining (J) or other domains of theimmunoglobulins, nor are disulfide bonds shown. However, where suchdomains are required for binding activity, they shall be constructed tobe present in the ordinary locations which they occupy in theimmunoglobulin molecules.

Alternatively, the adhesin sequences can be inserted betweenimmunoglobulin heavy chain and light chain sequences, such that animmunoglobulin comprising a chimeric heavy chain is obtained. In thisembodiment, the adhesin sequences are fused to the 3′ end of animmunoglobulin heavy chain in each arm of an immunoglobulin, eitherbetween the hinge and the C_(H)2 domain, or between the C_(H)2 andC_(H)3 domains. Similar constructs have been reported by Hoogenboom, etal., Mol. Immunol. 28:1027-1037 (1991).

Although the presence of an immunoglobulin light chain is not requiredin the immunoadhesins of the present invention, an immunoglobulin lightchain might be present either covalently associated to anadhesin-immunoglobulin heavy chain fusion polypeptide, or directly fusedto the adhesin. In the former case, DNA encoding an immunoglobulin lightchain is typically coexpressed with the DNA encoding theadhesin-immunoglobulin heavy chain fusion protein. Upon secretion, thehybrid heavy chain and the light chain will be covalently associated toprovide an immunoglobulin-like structure comprising two disulfide-linkedimmunoglobulin heavy chain-light chain pairs. Methods suitable for thepreparation of such structures are, for example, disclosed in U.S. Pat.No. 4,816,567, issued 28 Mar. 1989.

Immunoadhesins are most conveniently constructed by fusing the cDNAsequence encoding the adhesin portion in-frame to an immunoglobulin cDNAsequence. However, fusion to genomic immunoglobulin fragments can alsobe used (see, e.g. Aruffo et al., Cell 61:1303-1313 (1990); andStamenkovic et al., Cell 66:1133-1144 (1991)). The latter type of fusionrequires the presence of Ig regulatory sequences for expression. cDNAsencoding IgG heavy-chain constant regions can be isolated based onpublished sequences from cDNA libraries derived from spleen orperipheral blood lymphocytes, by hybridization or by polymerase chainreaction (PCR) techniques. The cDNAs encoding the “adhesin” and theimmunoglobulin parts of the immunoadhesin are inserted in tandem into aplasmid vector that directs efficient expression in the chosen hostcells.

4. Other C_(H)2/C_(H)3 Region-Containing Proteins

In other embodiments, the protein to be purified is one which is fusedto, or conjugated with, a C_(H)2/C_(H)3 region. Such fusion proteins maybe produced so as to increase the serum half-life of the protein.Examples of biologically important proteins which can be conjugated thisway include renin; a growth hormone, including human growth hormone andbovine growth hormone; growth hormone releasing factor; parathyroidhormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin;insulin A-chain; insulin B-chain; proinsulin; follicle stimulatinghormone; calcitonin; luteinizing hormone; glucagon; clotting factorssuch as factor VIIIC, factor IX, tissue factor, and von Willebrandsfactor; anti-clotting factors such as Protein C; atrial natriureticfactor; lung surfactant; a plasminogen activator, such as urokinase orhuman urine or tissue-type plasminogen activator (t-PA); bombesin;thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and-beta; enkephalinase; RANTES (regulated on activation normally T-cellexpressed and secreted); human macrophage inflammatory protein(MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors; ProteinA or D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4,NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, orTGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II);des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor bindingproteins; CD proteins such as CD3, CD4, CD8, CD19, CD20, CD34, and CD40;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments of any of the above-listed polypeptides.

The following examples are offered by way of illustration and not by wayof limitation. The disclosures of all citations in the specification areexpressly incorporated herein by reference.

EXAMPLES

The examples are provided so as to provide those of ordinary skill inthe art with a complete disclosure and description of how to make anduse the compounds, compositions, and methods of the invention and arenot intended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to insure accuracy with respect tonumbers used (e.g. amounts, temperature, etc.) but has some experimentalerrors and deviation should be accounted for. The disclosures of allcitations in the specification are expressly incorporated herein byreference.

For ease of reading, a list of abbreviations frequently used throughoutthe examples is provided below:

C_(f) Concentration in the filtrate (g/l)

C_(b) Bulk concentration (or feed concentration) (g/l)

CHO Chinese hamster ovary

CHOP Chinese hamster ovary cell protein(s)

CV Column volumes

DF Diafiltration

HCCF Harvested cell culture fluid

HCI Hydrophobic charge induction chromatography

HCP Host cell protein(s)

HIC Hydrophobic interaction chromatography

HPLC High performance liquid chromatography

HPTFF High performance tangential flow filtration

J Filtrate flux (lm⁻²h⁻¹)

Lp Membrane permeability

N Number of diavolumes

PHCP Purification factor based on HCP removal

PCHOP Purification factor based on CHOP removal

pI Isoelectric point

rhuMAb Recombinant humanized monoclonal antibody

S_(i) Sieving of solute “i”

Y Yield

ψ Selectivity

Example 1 Two Steps of Non-Affinity Purification

In the present example, the purification of anti-CD11a rhuMAb HCCF wasperformed with processes consisting of either two steps of non-affinitypurification or three steps of non-affinity purification using differentcombinations of non-affinity purification matrices.

The purification performance of cation exchange (such as by using an Scolumn), anion exchange (such as by using a Q column), mixed-mode ionexchange (such as by using ABx), hydroxyapatite (HA), hydrophobicinteraction (HIC) and hydrophobic charge induction (HCI) resins wereexamined in each step of the chromatographic purification process forthe anti-CD11a rhuMAb protein. Total host cell protein (CHOP) impurityremoval and protein yield was determined as described in detail inExample 2 and compared to traditional processes consisting of either twoor three steps and incorporating Protein A chromatography (i.e. for twostep processes, ProA followed by anion exchange (such as ProA-Q), andfor three steps processes, ProA followed by cation exchange, then anionexchange, such as by ProA-S-Q).

SP-SEPHAROSE FAST FLOW™ resin (S, cation exchange resin, AmershamBiosciences, Piscataway, N.J.), Q-Sepharose Fast Flow™ resin (Q, anionexchange resin, Amersham Biosciences, supra), Bakerbond ABx™ resin (ABx,mixed-mode ion exchange resin, J.T. Baker, Inc., Phillipsburg, N.J.),PHENYL-SEPHAROSE FAST FLOW™ resin (HIC, hydrophobic interaction resin,Amersham Biosciences, supra), Macroprep Ceramic Hydroxyapatite resin(RA, hydroxyapatite resin, BioRad Laboratories, Hercules, Calif.), andMEP HYPERCEL™ resin (HCI, hydrophobic charge induction resin, INVITROGENLIFE TECHNOLOGIES™, LifeTechnologies, Inc., Rockville, Md.) were eachpacked into 0.66 cm e.d.×20 cm Omni glass columns. The operatingconditions for chromatography are presented in Table 1. TABLE 1Chromatography Operating Conditions Mode of Load Resin Resin TypeOperation Buffers Conditioning SP Sepharose Fast Cation Non-specific 20mM MES, 50 mM <5 mS/cm Flow ™ (Amersham exchange bind and NaCl, pH 5.5pH 5.5 Biosciences, (S) elute 10 CV gradient to 500 mM Piscataway, NJ)NaCl Bakerbond ABx ™ Mixed-mode Non-specific Same as S Same as S (J. T.Baker, ion exchange bind and Phillipsburg, NJ) (ABx) elute Q SepharoseFast Anion Flow-through 25 mM Tris, 50 mM <7 mS/cm Flow ™ (Amershamexchange NaCl, pH 8 pH 8 Biosciences, NJ) (Q) Phenyl SepharoseHydrophobic Non-specific 50 mM MES, 0.8 M 0.8 M Na₂SO₄ Fast Flow ™, lowinteraction bind and Na₂SO₄, pH 6 pH 6 sub (Amersham (HIC) elute 15 CVgradient to 50 mM Biosciences, NJ) MES, pH 6 Macro-Prep HydroxyapatiteNon-specific 10 mM sodium <3 mS/cm ceramic (HA) bind and phosphate, pH6.8 pH 6.8 hydroxyapatite, elute 10 CV gradient to 400 mM Type II(Bio-Rad, phosphate, pH 6.8 Hercules, CA) MEP Hydrophobic Non-specific25 mM Tris, 50 mM pH > 7 HYPERCEL ™ charge bind and NaCl, 5 mM EDTA,(Life Technologies, induction elute pH 7.1, Rockville, MD) (HCI) Stepelute with 50 mM acetate, pH 4

All of the columns were loaded to 10 mg antibody per ml resin at a flowrate of 100 cm/h (5 column volumes per hour).

Between uses, S, HIC and HCI resins were sanitized with >3 columnvolumes of 0.5N NaOH. Columns containing ABx, Q, and HA resins werepacked with fresh resin before each use.

CHO cells expressing anti-CD11a rhuMAb were cultured and a harvestedcell culture formulation containing the antibody was collected. Thecrude cell culture mixture contained approximately 220,000 ppm CHOP(equivalent to 220,000 ng CHOP/mg anti-CD11a rhuMAb). An aliquot of thecrude mixture was applied to each of the resins for Step 1 in Table 2.An aliquot of the eluate from Step 1 was then applied to each of thealternative resins in Step 2 of Table 2, and antibody and impuritieswere further separated. The buffer conditions for each step aresummarized in Table 1. The crude mixture and each eluant pool from thefirst step were adjusted to the pH and ionic strength of the bufferconditions of the resin to which the crude mixture or eluate pool wasapplied for the subsequent purification step. A summary of purificationresults as measured by CHOP concentrations after each of two steps ofnon-affinity purification is shown in Table 2. TABLE 2 CHOP removal overtwo steps of non-affinity purification Resin CHOP Alternative CHOP usedin (ppm; ng/mg Resins used in (ppm; ng/mg Step 1 antibody) Step 2antibody) Test processes: Q 23,000 Q 14,000 HIC 3,000 ABx 1,000 S 900HCI 11,000 HIC 26,000 Q 9,900 HIC 2,400 ABx 400 S 900 Abx 6,600 Q 3,100HIC 2,400 ABx 1,700 S 1,000 HCI 2,800 S 14,000 Q 80 HIC 600 ABx 140 S2,100 Control process: ProA 300 S 30

Of the non-affinity steps examined, the ABx column removed the most CHOPimpurities from the HCCF, resulting in a CHOP concentration of 6600 ppm.The purity of pools resulting from two steps of non-affinitypurification, ranged from 80 ppm to 14,000 ppm CHOP. The purificationwith S purification as the first step and Q purification as the secondstep resulted in a low CHOP concentration of 80 ppm.

However, when the steps were reversed such that Q purification was thefirst step and S purification was the second step; the purificationyield was a CHOP concentration of 900 ppm. The step order of thenon-affinity processes affected the purity results.

Further purification using three steps of non-affinity purification wasevaluated and compared to a three step purification process involvingone affinity step of Protein A chromatography, i.e. ProA-S-Q, as shownin Tables 3 and 4. As for the studies described above for a 2-steppurification process, aliquots of a crude cell culture mixturecontaining 220,000 ppm CHOP were adjusted for pH and ionic strengthaccording to Table 1 for the resin to which they were applied in Step 1of Table 3, and similarly for Steps 2 and 3 of Table 3. The results ofCHOP removal using processes involving three steps of non-affinitypurification are shown in Table 3. The yields of anti-CD11a rhuMAb fromsome of the three-step non-affinity purification processes are shown inTable 4. TABLE 3 CHOP removal over three steps of non-affinitypurification Resin [CHOP], [CHOP], Alternative [CHOP], used in ppm,after Resin used ppm, after Resins used ppm, after Step 1 Step 1 in Step2 Step 2 in Step 3 Step 3 Test Processes: HIC 26,000 ABx 400 ABx 13 S 14HIC 20 Q 22 S 14,000 Q 80 ABx <2 S 10 HIC <2 Q 30 S 14,000 ABx 140 ABx28 S 50 HIC 6 Q <2 Control process: ProA 730 S 160 Q <2

TABLE 4 Yields of anti-CD11a rhuMAb over three steps of non-affinitypurification Process Steps Step 1 Step 2 Step 3 Overall Yield Testprocesses: S Q ABx 76% 96% 100% 79% S Q HIC 85% 96% 100% 89% S ABx Q 88%96%  92% 100%  Control process: ProA S Q 85% 97%  89% 98%

The combination of two or more steps of non-affinity purificationresulted in a purity level, as determined by the elimination of CHOPimpurities, of about, for example, 14,000 ppm of CHOP after the firstnon-affinity step, about 80 ppm of CHOP after the second non-affinitystep and about 2 ppm of CHOP after the third step of the process(S-Q-ABx or HIC) (see Table 2), and product purities of anti-CD11aantibody as shown in Table 4.

Example 2 Combination of Non-Affinity Chromatography and HPTFFPurification

The present example involves the purification of recombinant humanmonoclonal antibody, anti-HER2 rhuMAb, with a molecular weight of 160 kDand a pI of about 9.0 from chinese hamster ovary (CHO) cells. Theanti-HER2 rhuMAb was obtained from an industrial scale CHO cell cultureprocess at Genentech (South San Francisco, Calif., USA). After CHO cellculture, the anti-HER2 rhuMAb molecule was partially clarified bycentrifugation and normal cell filtration to remove cells and celldebris. The resulting pool consisted of 0.52 mg/ml of anti-HER2 rhuMAbproduct and 0.78 mg/ml of CHOP.

For purification of anti-HER2 rhuMAb, conditioned harvested cell culturefluid (HCCF) comprising an anti-HER2 rhuMAb product and Chinese HamsterOvary host cell proteins (CHOP) from CHO cells expressing anti-HER2rhuMAb was loaded onto an initial cation exchange chromatography column(S) (SP-SEPHAROSE FAST FLOW™ Resin, Amersham Biosciences) to remove hostcell proteins or CHO proteins (CHOP), variants, and aggregates. Elutionsfrom the S column were pooled (S pool) and subjected to a second anionexchange chromatography column (O) (Q-SEPHAROSE FAST FLOW™ resin,Amersham Biosciences, Piscataway, N.J.) to remove CHOP and targetprotein aggregates. The flow-through from the Q column (Q pool) wassubdivided and each pool was further subjected to a third process ofHPTFF for further removal of CHOP, variants and small molecules. Two ofthe Q pools were subjected to HPTFF Experiment 1 and HPTFF Experiment 2as described in detail below.

A. Non-Affinity Chromatography

The chromatography columns were loaded to approximately 10 grams ofrhuMAb/liter of resin for a total of about 40 grams of rhuMAb at a flowrate of 100 cm/h

(5 column volumes (CVs) per hour).

1. Methods

For preparation of the non-affinity chromatography columns,bind-and-elute SP-Sepharose and flow-through Q-Sepharose were eachpacked into preparative scale columns. The operating conditions for eachchromatography column are presented in Table 5. TABLE 5 Non-affinityChromatography Operating Conditions Resin Mode of Load Resin TypeOperation Buffers Conditioning SP Sepharose Fast Cation exchangeNon-specific 25 mM MES, 20 mM NaCl, <6 mS/cm Flow ™ (Amersham (S) bindand pH 5.5 pH 5.5 Biosciences, elute 10 CV gradient to 500 mMPiscataway, NJ) NaCl Q Sepharose Fast Anion exchange Flow-through 25 mMTris, 50 mM NaCl, <8 mS/cm Flow ™ (Amersham (Q) pH 8 pH 8 Biosciences,NJ)

The HCCF was conditioned by diluting the HCCF to a conductivity of lessthan 6 mS/cm with water and adjusting the HCCF to a pH of 5.5 with HCland filtered through a 0.22 μm filter. A volume of 66 liters ofconditioned HCCF was subjected to non-affinity chromatography.

The SP-Sepharose column was equilibrated with 5 column volumes (CVs) ofthe column buffer (Table 5). The 66 liters of conditioned HCCF (<10 g/l)were loaded onto the equilibrated SP-Sepharose column. After loading theconditioned HCCF onto the SP-Sepharose column, the column was washedwith 5 CVs of column buffer. Elutions were made with elution buffer (25mM MES, 500 mM NaCl, pH 5.5) with eluant collected at an absorbance offrom 0.1-0.2 AU at 280 nm. The chromatography resin was regenerated in a0.5 M NaOH solution and further stored in 0.1 M NaOH.

The collections from the SP-Sepharose column were pooled (SP pool) andconditioned by diluting the S pool to a conductivity of approximately7.5 to 8 mS/cm with water and adjusted to a pH of 8 with NaOH. Theconditioned S pool was then filtered through a 0.22 μm filter. Thefiltered SP pool (about 9 liters) was loaded onto a Q-Sepharose columnthat was equilibrated with 5 CVs of the column buffer (see Table 5). Theflow-through was collected at 0.2-0.2 AU at 280 nm and the flow-throughwas pooled (Q pool). A total of 20.6 liters of the Q pool was collected.

The Q-Sepharose chromatography resin was regenerated in a 0.5 M NaOHsolution and further stored in 0.1 M NaOH. The 20.6 liters recoveredfrom the Q column was divided into 3 identical pools, each having avolume of 6.9 liters and a concentration of about 1.4 g/L of anti-HER2rhuMAb, prior to HPTFF purification.

2. Analysis

The amount of anti-HER2 rhuMAb in each pool following a purificationstep of the process, i.e. in the HCCF and in the pools from thepurification process, was determined by an HPLC analysis based onProtein-A immunoaffinity. The HPLC column was a Poros Protein A, 4.6 mmi.d.×100 mm bed height (PerSeptive Biosystems). Samples and standardswere applied to the column in a loading buffer, the rhuMAb analyte boundto the column, then was eluted under acidic conditions. The peak area ofthe eluted material was compared to the peak area of a standard curve tocalculate the amount of rhuMAb. The assay range was typically 0.05 mg/mLto 1.0 mg/mL.

Upon completion of the S and Q chromatography, samples from pools weresubjected to SDS-PAGE analysis (FIG. 2, lanes 4 and 5, respectively).Samples were analyzed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE), which separated proteins according to size(relative hydrodynamic radius). Samples and the molecular weightstandard (ranging from 10 to 200 kDa) were prepared under non-reducingconditions and loaded onto a gel at approximately 2.5 μg/lane. A 10% to20% acrylamide gradient gel, 8 cm×8 cm size, was used herein (Z-axisInternational, Inc., Hudson, Ohio) and was electrophoresed at a constantvoltage of 170 mV. Following the electrophoresis, the proteins werestained to be rendered visible. The electrophoresed gel was then treatedby silver staining according to the method described by Morrisey(Morrisey, J., Analytical Biochemistry, 1981, 117: 307-310). The resultsare shown in FIG. 2.

To determine the amount of anti-HER2 rhuMAb present in each pool as theintact monomer, the mixtures were subjected to Size ExclusionChromatography (SEC) according to the following procedure. Briefly, aSuperdex 200 HR 10/30 column (Amersham Biosciences, Piscataway, N.J.)was equilibrated in phosphate buffer saline. Approximately 100 μg ofrhuMAb per sample was applied to the column. The sample was eluted fromthe column based on the molecular size of the protein moleculescontained in the sample (optimal separation range: 10 to 600 kDa). Theabsorbance of the column eluate was measured at 280 nm and the proteinelution peaks were integrated to determine the percent area of monomericrhuMAb. The percentages of intact monomer anti-HER2 rhuMAb in the HCCF,S pool, Q pool, and HPTFF pool are shown in Table 8.

CHOP concentration was determined by an enzyme-linked immunosorbent(ELISA) assay using goat anti-(host-cell protein) antibodies to quantifyCHOP. Affinity-purified goat whole anti-CHOP antibodies were immobilizedon microtiter plate wells. Dilutions of the pool samples, containingCHOP, were incubated in the wells, followed by incubation withconjugated-peroxidase whole anti-CHOP. The horseradish peroxidase wasthen quantified with o-phenylenediamine by reading the absorbance at 492nm. Based on the principle of sandwich ELISA, the concentration ofperoxidase corresponded to the CHOP concentration. The assay range forthe ELISA was typically 5 ng/mL to 320 ng/mL. Depending on theconcentration of the samples, 2 to 4 dilutions per sample were assayedand the dilution-corrected results were averaged.

B. HPTFF

High Performance Tangential Flow Filtration (HPTFF) is a two-dimensionalfiltration operation that involves separation of solutes with less thana 10-fold size difference based on both size and charge. As mentionedabove, the Q pool was divided into three equivalent Q pools, each with avolume of 6.9 L and a concentration of about 1.4 g/L of anti-HER2rhuMAb, prior to further purification by HPTFF. Two of the Q pools weresubjected to HPTFF Experiment 1, each involving different conditions.Upon determination of the optimum conditions for HPTFF from the HPTFFExperiment 1 studies, combined pools from HPTFF Experiments 1 and 2 weresubjected to additional HPTFF as described in detail below.

1. Membrane

Filtration membranes used for HPTFF in these Examples are CompositeRegenerated Cellulose (CRC)-Millipore ULTRACEL™ (Millipore) with anominal molecular weight cut-off of 300 kD (PLCMK). CRC300mini-PELLICON2® membranes (Millipore) was charge-modified as describedherein, resulting in the charged cellulose membrane CRC300+ used in theHPTFF studies of this Example. Briefly, a 300 kD PELLICON-2® cassette(membrane area of 0.1 m²) was used for the scale-down experiment(minimum of 1 L solution). The membrane was cleaned according to acartridge preparation protocol before the first use to remove anyresidual storage and shipping solution and to equilibrate the membraneto the appropriate buffer condition. The membrane was chemicallymodified in situ using bromo-propyl-trimethyl-ammonium bromide(Sigma-Aldrich, St Louis, Mo.) under alkaline conditions(PCT/US00/19964, the entire contents of which is hereby incorporated byreference). Specifically, the membrane was charged without co-currentfiltrate flow, at constant filtrate flux of 100 lm⁻²h⁻¹, retentatepressure fixed at 10 psig, total recycle with filtrate open mode with 1L of ligand dissolved in 0.1N NaOH and 0.2 μm filtered. The Lp beforecharging was about 53 lm⁻²h⁻¹/psi in 0.1N NaOH, and the Lp aftercharging was about 37 lm⁻²h⁻¹/psi in 0.1N NaOH. After charging, theresulting positively charged membrane was cleaned using 0.1N sodiumhydroxide, sanitized with 300 ppm of MINNCARE™ solution, and stored in0.1N sodium hydroxide. Before each HPTFF experiment, the membrane wasflushed with the first diafiltration buffer of the experiment to removestorage solution and was tested for integrity. Membrane permeability wasmeasured using the HPTFF system with co-current filtrate flow at aminimum of three filtrate fluxes.

2. HPTFF Filtration System

HPTFF experiments were performed using a fully automated tangential flowfiltration system with the basic configuration illustrated in FIG. 1.The HPTFF system included a 40-liter stainless steel recycle tank, feedand filtrate flow meters (Admag Model 102 and 105, Johnson YokogawaCorp., Newman, Ga.) and pressure transducers (Model MSP220-A2, 0-100psig=0-7 bar, Anderson Instruments, Fultonville, N.Y.). The feed andco-current filtrate flow pumps were positive displacement pumps(Universal 6, Waukesha-Cherry Burrell, Delavan, Wis.) while thediafiltration and filtrate pumps were peristaltic pumps (ModelL-7518-62, Cole Parmer, Niles, Ill.). The recycle tank included atemperature probe (Model RIX, −29° C. to 82° C., Moore Industries,Sepulveda, Calif.). All piping was constructed of 316L stainless steel.The retentate pressure control valve was actuated using a steeldiaphragm (Model 1/2 Mikroseal packless control valve, H.D. Baumann,Portsmouth, N.H.), while all other valves were pneumatically actuatedwith ethylene propylene diene monomer diaphragms (Biotek Model8836-18-BH, ITT Sherotec, Simi Valley, Calif.). Continuous tank liquidlevel was measured with a magneto restrictive probe (Model TempsonicsII, MTS, Research Triangle Park, N.C.). Data acquisition and controlwere performed using proprietary software (Genentech, Inc., South SanFrancisco, Calif.) using a MycroAdvantage software shell (MooreProducts, Springhouse, Pa.).

HPTFF was conducted at a fixed feed flow rate of 323 l.m⁻².h⁻¹(volumetric feed flow rate divided by the membrane area) and a retentatepressure of 10 psi. The co-current filtrate flow rate was controlled toreach equal transmembrane pressure at the inlet (feed) and outlet(retentate) of the membrane cassette. The filtrate flux was set at 50l.m⁻².h⁻¹ by adjusting the filtrate pump rate. The start-up of HPTFFexperiments included a ramp-up of all flow rates in order to minimizethe difference between transmembrane pressure at the inlet and outlet ofthe cassette. The retentate was recycled to the feed tank, while thefiltrate was directed to a collection vessel. Feed and filtrate sampleswere collected in both cases for product and HCP analysis.

a. HPTFF Experiment 1

After the division of the Q pool, as described above, into equivalent6.9 L Q pools, one of the Q pools was subjected to HPTFF Experiment 1using a CRC300+membrane under the following conditions.

The charged membranes were equilibrated in the first diafiltrationbuffer for this experiment (see Table 7). The Q pool was diluted tolower the ionic strength and conductivity of the rhuMAb pool to 2.7mS/cm, adjusted to a pH of 4.5 and then added to the feed tank (FIG. 1).The material in the feed tank was subjected to concentration by removalof a portion of the solution. When the bulk volume reached a bulkconcentration (Cb) of 10 g/L, the solution in the feed tank wassubjected to sequential diafiltration steps. With a constantconductivity of 1.5 mS/cm, diafiltration was performed with 10diavolumes at a pH of 4.5 and 5 diavolumes each of pH 5.0, pH 5.5, pH6.0, and pH 6.5 (Table 7). The yield was calculated based on thequantifiable product sieving during diafiltration using the followingequation: Y=e^(−NS) ^(T arg etprotein) where S is the sieving of thetarget protein and N the number of diavolumes. TABLE 7 Experimentalconditions and results from HPTFF step of purification Concen- trationDiafiltration Yield [CHOP]_(final) C_(b) (g/L) pH - N (%) ppm Q pool 410HPTFF Experiment 1 10 4.5 - 10 99% 21 5.0 - 5 5.5 - 5 6.0 - 5 6.5 - 5HPTFF Experiment 2 10 4.5 - 10 99% 17 5.5 - 10 6.5 - 10 Additional HPTFF10 6.5 - 40 99% 2.2 (Combined pools from 6.0 - 5 HPTFF 1 & 2)

The product quality of the recovered pool from the HPTFF Experiment 1was subjected to analysis, including SDS-PAGE gel electrophoresis (FIG.2, lanes 10), rhuMAb % intact monomer analysis and CHOP concentrationanalysis (Table 8), as described above.

Significant sieving of CHOP was observed with CRC300+without anysignificant loss of anti-HER2 rhuMAb. The final concentration of CHOP inthe recovered pool from HPTFF performed with CRC300+was 21 ppm.

b. HPTFF Experiment 2

Another of the Q pools, as described above, having 1.4 mg/ml of rhuMAb,410 ppm of CHOP, a pH of 5.6 and a conductivity of about 8 mS/cm, wassubjected to a HPTFF Experiment 2.

As described above, the CRC300+membrane was equilibrated in the firstdiafiltration buffer for this HPTFF Experiment 2 (see Table 7). The Qpool was diluted with water to lower the conductivity to 2.4 mS/cm. ThepH was adjusted to pH 4.5 with HCl. The resulting conditioned pool wasloaded onto the feed tank. In a single operation, the conditioned poolwas then concentrated to 10 g/L at pH 4.5, followed by a constantretentate volume diafiltration comprising a specific sequence ofdiafiltration buffers (Table 7). Three sequences of diafiltrationbuffers were selected as follows: 10 diavolumes each at a pH of 4.5,5.5, and 6.5, at a constant conductivity of 1.5 mS/cm. All HPTFFexperiments were conducted at a filtrate flux of 50 l.m⁻².h⁻¹ using apositively charged Pellicon-2® mini cassette with a permeability of 36l.m⁻².h⁻¹/psi.

Upon completion of the HPTFF process performed with CRC300+, a sample ofthe recovered pool was subjected to SDS-PAGE analysis (FIG. 2, lane 11).The product quality of the recovered pool from the HPTFF Experiment 2was subjected to additional analysis, including Size ExclusionChromatography (SEC) and CHOP concentration analysis (Table 8).

The purification factors for the HPTFF step was greater than 24 (i.e.24-fold removal of CHOP) and CHOP removal occurred during bothconcentration and diafiltration. The CHOP concentration was reduced from410 ppm (concentration in the material recovered from the Qchromatography column) to 17 ppm (concentration in the materialrecovered from the HPTFF Experiment 2) (see Table 8). No significantfiltrate losses were observed. TABLE 8 CHO host cell proteinquantification and purity analysis of anti-HER2 rhuMAb feedstream inpurification processes % intact rhuMAb monomer Purification Step [CHOP](ppm) (measured by SEC) HCCF 1,469,000 — S pool 144,780 95.9% Q pool 41097.4% HPTFF Experiments 1, 2 21, 17 99.8% Control Process: <1  100%(using steps: ProA-S-Q-UFDF)

c. Additional HPTFF

Material recovered after the HPTFF Experiments 1 and 2 was combined andfurther subjected to additional HPTFF as follows.

As described above, the CRC300+membrane was equilibrated in the firstdiafiltration buffer and the combined material was loaded onto the feedtank. The material in the feed tank was subjected to optimal sequentialdiafiltration steps as follows: 40 diavolumes at pH 6.5 and 1.5 mS/cm,followed by 5 diavolumes at pH 6.0 and 0.3 mS/cm.

The additional HPTFF process reduced the concentration of CHOP in thefinal retentate to 2.2 ppm. A sample of the recovered material after theadditional was subjected to SDS-PAGE analysis. The product quality ofthe material recovered after additional HPTFF (FIG. 2, lanes 6 and 12)as determined by SDS-PAGE analysis was compared to the product qualityof material obtained through a conventional purification processinvolving ProA, SP, Q and UFDF (FIG. 2, lane 7).

The purification process, involving two steps of non-affinitypurification and a third step of HPTFF, resulted in a purity level, asdetermined by the elimination of CHOP impurities, of about 144,780 ppmof CHOP after S purification, of about 410 ppm CHOP after Qpurification, and a final purity of about 17-21 ppm of CHOP. Furtherpurity of about 2.2 ppm of CHOP was achieved by additional HPTFF, whichfurther purification is alternatively incorporated into the third step,thereby providing a three-step non-affinity process comparable totraditional methods using costly affinity chromatography.

Example 3 Combination of Non-Affinity Chromatograph and HPTFFPurification

The present example involves the purification of recombinant humanmonoclonal antibody, anti-CD40 rhuMAb, with a molecular weight of 160 kDand a pI of about 9.3 from chinese hamster ovary (CHO) cells. Theanti-CD40 rhuMAb was obtained from an industrial scale CHO cell cultureprocess at Genentech (South San Francisco, Calif., USA). After CHO cellculture, the anti-CD40 rhuMAb molecule was partially clarified bycentrifugation and normal cell filtration to remove cells and celldebris. The resulting pool consisted of 1.7 mg/ml of anti-CD40 rhuMAbproduct and approximately 0.4 mg/ml of CHOP.

For purification of anti-CD40 rhuMAb, conditioned harvested cell culturefluid (HCCF) comprising an anti-CD40 rhuMAb product and Chinese HamsterOvary host cell proteins (CHOP) from CHO cells expressing anti-CD40rhuMAb was loaded onto an initial cation exchange chromatography column(S) (SP-SEPHAROSE FAST FLOW™ Resin, Amersham Biosciences) to remove hostcell proteins or CHO proteins (CHOP), variants, DNA impurities andaggregates. Elutions from the S column were pooled (S pool) andsubjected to a second anion exchange chromatography column (O)(Q-SEPHAROSE FAST FLOW™ resin, Amersham Biosciences, Piscataway, N.J.)to remove CHOP, DNA impurities and target protein aggregates. Theflow-through from the Q column (Q pool) was further subjected to a thirdprocess of HPTFF for further removal of CHOP, variants and smallmolecules.

A. Non-Affinity Chromatography

1. Methods

For preparation of the non-affinity chromatography columns,bind-and-elute SP-Sepharose and flow-through Q-Sepharose were eachpacked into preparative scale columns. The operating conditions for eachchromatography column are presented in Table 9. TABLE 9 Non-affinityChromatography Operating Conditions Mode of Load Resin Resin TypeOperation Column Buffers Conditioning SP Sepharose Fast Cation exchangeNon-specific 20 mM MES, 50 mM <7.0 mS/cm Flow ™ (Amersham (S) bind andNaAcetate, pH 6.5 pH 6.5 Biosciences, elute Piscataway, NJ) Q SepharoseFast Anion exchange Flow-through 25 mM Tris, 50 mM NaCl, <8 mS/cm Flow ™(Amersham (Q) pH 8 pH 8 Biosciences, NJ)

The HCCF was conditioned by diluting the HCCF to a conductivity of lessthan 7 mS/cm with water and adjusting the HCCF to a pH of 6.5 withacetic acid and filtered through a 0.22 μm filter. The SP-Sepharosecolumn was equilibrated with 4 column volumes (CVs) of the column buffer(Table 9) and loaded to approximately 30 grams of rhuMAb/liter of resinfor a total of about 13 grams of rhuMAb at a flow rate of 150 cm/h.After loading the conditioned HCCF onto the SP-Sepharose column, thecolumn was washed with 5 CVs of wash buffer (20 mM HEPES, 35 mMNaAcetate, pH 8.0) followed by 3 CVs of column buffer (Table 9).Elutions were made with a 10 CV gradient elution from the column bufferto the elution buffer 20 mM MES, 140 mM NaAcetate, pH 6.5, with theeluant collected at an absorbance of from 0.1 to 0.5 AU at 280 nm. Thechromatography resin was regenerated in a 0.5 M NaOH solution andfurther stored in 0.1 MNaOH.

The SP-Sepharose pool (SP pool) was conditioned by diluting the S poolto a conductivity of approximately 7.5 mS/cm with water and adjusted toa pH of 8 with NaOH. The conditioned S pool, having a total mass ofabout 9 grams, was then filtered through a 0.22 μm filter. The filteredconditioned SP pool was loaded onto a Q-Sepharose column that wasequilibrated with 5 CVs of the column buffer (see Table 9). Theflow-through was collected at 0.2-0.2 AU at 280 nm and the flow-throughwas pooled (Q pool). The Q-Sepharose chromatography resin wasregenerated in a 0.5 M NaOH solution and further stored in 0.1 M NaOH.

2. Analysis

The amount of anti-CD40 rhuMAb in each pool following a purificationstep of the process, i.e. in the HCCF and in the pools from thepurification process, was determined by an HPLC analysis based onProtein-A immunoaffinity as described in the Example 2 herein. CHOPconcentration was determined using the enzyme-linked immunosorbent(ELISA) assay described in the Example 2 herein. Upon completion of theS and Q chromatography, samples from pools were subjected to SDS-PAGEanalysis (FIG. 5, lanes 3 and 4, respectively). The Q pool was dilutedto lower the ionic strength and conductivity of the rhuMAb pool to 1.8mS/cm, adjusted to a pH of 4.5 and then added to the recycle tank (FIG.1). The HPTFF experiment purification step using a positively chargedCRC300+membrane (the HPTFF experiment) was begun by first concentratingthe material from the Q pool until the bulk volume reached a bulkconcentration (Cb) of 10 g/L. The resultant solution in the recycle tankwas then subjected to sequential diafiltration steps. With a constantconductivity of 1.5 mS/cm, diafiltration was performed with 5 diavolumeseach at a pH 4.5 and pH 5.5, followed by 20 diavolumes at pH 6.5,followed by 10 diavolumes at pH 7.0 (Table 10). The yield was calculatedbased on the quantifiable product sieving during diafiltration using thefollowing equation: Y=e^(−NST arg etprotein) where S is the sieving ofthe target protein and N the number of diavolumes. TABLE 10 Experimentalconditions and results from HPTFF step of purification ConcentrationDiafiltration Yield [CHOP] [DNA] C_(b) (g/L) pH - N (%) (ppm) (ppm) Qpool 96% 15 15 HPTFF pool 10 4.5-5  99% <0.6 <0.6 5.5-5  6.5-20 7.0-10

The product quality of the recovered pool from this HPTFF experiment wassubjected to analysis including SDS-PAGE gel electrophoresis (FIG. 5,lane 5), rhuMAb % intact monomer analysis, and CHOP concentrationanalysis (Table 11), as described in Example 2, herein. DNAconcentration was evaluated according to the THRESHOLD® Total DNA Assay(Molecular Devices, Corp., Sunnyvale, Calif.) (Table 11). The THRESHOLD®Total DNA Assay is specific for single-stranded DNA, which is obtainedfrom the sample via denaturation by heat. The single-stranded DNA islabeled with binding proteins, which are covalently bound to urease andstreptavidin, and form a DNA complex. The DNA complex is filteredthrough a biotin coated nitrocellulose membrane known as a “stick.” Thebiotin on the membrane reacts with streptavidin in the DNA complex,capturing the complex. The stick is placed in the Threshold Reader,which contains the substrate, urea. The enzymatic reaction between ureaand urease (in the DNA complex) changes the local pH of the substratesolution. A silicon sensor records a change in surface potential, whichis proportional to the pH change. The rate of change in surfacepotential is proportional to the amount of DNA. Quantification ofsamples is determined by comparison to DNA standards. Samples werediluted so that the DNA content falls within the reporting range of thestandard curve (10-400 pg/mL).

Significant sieving of CHOP was observed with positively chargedCRC300+HPTFF membrane without any significant loss of positively chargedanti-CD40 rhuMAb. CHOP removal occurred during both concentration anddiafiltration. The CHOP concentration was reduced from 15 ppm(concentration in the material recovered from the Q chromatographycolumn) to less than 0.6 ppm within the first 20 diavolumes(concentration in the protein pool in the recycle tank). The removal ofCHOP impurities was confirmed by measuring the concentration in thematerial recovered from the HPTFF experiment (see Table 11). Nosignificant filtrate losses were observed. TABLE 11 CHO host cellprotein quantification and purity analysis of anti-CD40 rhuMAbfeedstream in purification processes % intact [CHOP] rhuMAb monomer[DNA] Purification Step (ppm) (measured by SEC) (ppm) HCCF 240,000— >5441 S pool 530 — 0.1 Q pool 15 — <0.01 HPTFF pool <0.6 99.5% <0.006Control Process: 3 99.5% <0.003 (using steps: ProA-S-Q- UFDF)

The purification process, involving two steps of non-affinitypurification and a third step of HPTFF, resulted in a purity level, asdetermined by (1) the elimination of CHOP impurities, of about 530 ppmof CHOP after S purification, of about 15 ppm CHOP after Q purification,and a final purity of about less than 0.6 ppm of CHOP within 20diavolumes, and by (2) the elimination of DNA impurities, of about 0.1ppm of CHOP after S purification, of about less than 0.01 ppm DNA afterQ purification, and a final purity of about less than 0.006 ppm of DNA.In addition, the electrophoresis analysis illustrated the comparablepurity of the non-affinity final pool (FIG. 5, lane 5) to that aconventional pool obtained using an affinity step (FIG. 5, lane 10).

FIG. 5 shows a silver-stained SDS-PAGE gel containing samples that weretaken at different points during the purification of anti-CD40recombinant human monoclonal antibody (rhuMAb) according to Example 3(lanes 2-5) and compared to a conventional purification processincluding an affinity purification step (lanes 8-10). The arrowsindicating 160 kD, 50 kD, and 25 kD point to the full length antibody,the heavy chain, and the light chain, respectively. Other bands areanti-CD40 rhuMAb fragments. Lane 1 is a mixture of protein standards.Lanes 2-6 are samples taken after performance of the non-affinityprocess disclosed in Example 3 herein in which host cell culture fluid(HCCF) (lane 2) was purified by cation exchange chromatography (S pool,lane 3), followed by an anion exchange chromatography (Q pool, lane 4),followed by HPTFF using a charged membrane (HPTFF pool, lane 5),followed by and compared to material recovered after rinsing the HPTFFmembrane and the feed side of the HPTFF apparatus (HPTFF buffer flushpool, lane 6). Lane 7 is blank. Lanes 8-10 correspond to anti-CD40 in anHCCF mixture purified by a conventional recovery process including aprotein A affinity chromatography step (not shown), followed by a cationexchange chromatography step (lane 8), followed by an anion exchangechromatography (lane 9), and followed by an ultrafiltration step (lane10).

This purification scheme provided a three-step non-affinity processcomparable to traditional methods using costly affinity chromatography.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the examples presentedherein, since the exemplified embodiments are intended as illustrationsof certain aspects of the invention and any functionally equivalentembodiments are within the scope of this invention. The examplespresented herein are not intended as limiting the scope of the claims tothe specific illustrations. Indeed, various modifications of theinvention, in addition to those shown and described herein and whichfall within the scope of the appended claims, may become apparent tothose skilled in the art from the foregoing description.

1. A method for purifying a target protein from a mixture containing ahost cell protein, comprising subjecting said mixture to: (a) anon-affinity purification step, followed by (b) high-performancetangential-flow filtration (HPTFF), and (c) isolating said protein in apurity containing less than 100 parts per million (ppm) of said hostcell protein, wherein said method includes no affinity purificationstep.
 2. A method for purifying a target protein from a mixturecontaining a host cell protein, comprising subjecting said mixture to:(a) a first non-affinity purification step, and (b) a secondnon-affinity purification step, followed by (c) high-performancetangential-flow filtration (HPTFF), and (d) isolating said protein in apurity containing less than 100 parts per million (ppm) of said hostcell protein, wherein said method includes no affinity purificationstep.
 3. The method of claim 2 wherein said first and secondnon-affinity purification steps are different and are selected from thegroup consisting of ion exchange chromatography and hydrophobicinteraction chromatography.
 4. The method of claim 3 wherein said ionexchange chromatography is selected from the group consisting of cationexchange chromatography, anion exchange chromatography and mixed modeion exchange chromatography.
 5. The method of claim 4 wherein said firstand second affinity purification steps are cation exchangechromatography and anion exchange chromatography, in either order. 6.The method of claim 4 wherein said first non-affinity purification stepis cation exchange chromatography and said second non-affinitypurification step is anion exchange chromatography.
 7. The method ofclaim 6 wherein said method consists of purification steps (a)-(c)followed by isolation step (d).
 8. The method of claim 6 wherein saidcation exchange chromatography step is performed on a cation exchangeligand selected from the group consisting of carboxy-methyl, BAKERBONDABX,™ sulphopropyl (SP), and sulphonyl.
 9. The method of claim 6 whereinsaid cation exchange chromatography step is performed on a cationexchange resin selected from the group consisting ofcarboxy-methyl-cellulose, BAKERBOND ABX,™ sulphopropyl immobilized onagarose, and sulphonyl immobilized on agarose.
 10. The method of claim 6wherein said anion exchange ligand is selected from the group consistingof DEAE and quaternary ammonium ions.
 11. The method of claim 6 whereinsaid anion exchange resin is selected from the group consisting of DEAEcellulose, QAE SEPHADEX™ and FAST SEPHAROSE.™
 12. The method of claim 1or 2 wherein the HPTFF is performed using a charged membrane.
 13. Themethod of claim 1 or 2 wherein said host cell protein is Chinese HamsterOvary Protein (CHOP).
 14. The method of claim 1 or 2 wherein said targetprotein is an antibody.
 15. The method of claim 14 wherein said antibodyis a monoclonal antibody.
 16. The method of claim 14 wherein saidantibody is a polyclonal antibody.
 17. The method of claim 14 whereinsaid antibody is a humanized antibody.
 18. The method of claim 14wherein said antibody is a human antibody.
 19. The method of claim 14wherein said antibody is an antibody fragment.
 20. The method of claim14 wherein said antibody fragment is selected from the group consistingof Fab, Fab′, F(ab′)₂ and Fv fragments, single-chain antibody molecules,diabodies, linear antibodies, bispecific antibodies and multispecificantibodies formed from antibody fragments.
 21. The method of claim 14wherein said antibody specifically binds to an antigen selected from thegroup consisting of CD3, CD4, CD8, CD19, CD20, CD34, CD40, EGF receptor,HER2, HER3, HER4 receptor, LFA-1, Mac1, p150,95, VLA-4, ICAM-1, VCAM,av/b3 integrin, CD11a, CD18, CD11b, VEGF, IgE, flk2/flt3 receptor,obesity (OB) receptor, mpl receptor, CTLA-4, and polypeptide C.
 22. Themethod of claim 14 wherein said antibody is selected from the groupconsisting of anti-HER2; anti-CD20; anti-IL-8; anti-VEGF; anti-PSCA;anti-CD11a; anti-IgE; anti-Apo-2 receptor; anti-TNF-α; anti-TissueFactor (TF); anti-CD3; anti-CD25; anti-CD34; anti-CD40; anti-tac;anti-CD4; anti-CD52; anti-Fc receptor; anti-carcinoembryonic antigen(CEA) antibodies; antibodies directed against breast epithelial cells;antibodies that bind to colon carcinoma cells; anti-CD33; anti-CD22;anti-EpCAM; anti-GpIIb/IIIa; anti-RSV; anti-CMV; anti-HIV;anti-hepatitis; anti-αvβ3; anti-human renal cell carcinoma; anti-human17-1A; anti-human colorectal tumor; anti-human melanoma; anti-humansquamous-cell carcinoma; and anti-human leukocyte antigen (HLA)antibodies.
 23. The method of claim 14 wherein said antibody is selectedfrom the group consisting of anti-HER2 receptor, anti-VEGF, anti-IgE,anti-CD20, anti-CD11a, and anti-CD40 antibodies.
 24. The method of claim1 or 2 wherein the target protein is an immunoadhesin.
 25. The method ofclaim 1 or 2 wherein the target protein is an antibody-like molecule.26. The method of claim 25 wherein said antibody-like molecule is aprotein fused to, or conjugated with, a C_(H)2/C_(H)3 region.
 27. Themethod of claim 26 wherein said protein is selected from the groupconsisting of renin; growth hormones; growth hormone releasing factor;parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;factor VIIIC; factor IX; tissue factor; von Willebrands factor; ProteinC; atrial natriuretic factor; lung surfactant; urokinase; human urineand tissue-type plasminogen activator (t-PA); bombesin; thrombin;hemopoietic growth factor; tumor necrosis factor-alpha and -beta;enkephalinase; RANTES; human macrophage inflammatory protein(MIP-1-alpha); serum albumins; Muellerian-inhibiting substance; relaxinA-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associatedpeptide; beta-lactamase; DNase; IgE; cytotoxic T-lymphocyte associatedantigens (CTLAs); inhibin; activin; vascular endothelial growth factor(VEGF); receptors for hormones or growth factors; Protein A or D;rheumatoid factors; bone-derived neurotrophic factor (BDNF);neurotrophin-3, -4, -5, and -6 (NT-3, NT-4, NT-5, and NT-6), nervegrowth factors; platelet-derived growth factor (PDGF); fibroblast growthfactors; epidermal growth factor (EGF); transforming growth factors(TGF); insulin-like growth factor-I and -II (IGF-I and IGF-II);des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor bindingproteins (IGFBPs); CD proteins; erythropoietin; osteoinductive factors;immunotoxins; bone morphogenetic proteins (BMPs); interferons-alpha,-beta, and -gamma; colony stimulating factors (CSFs); interleukins IL-1to IL-10; superoxide dismutase; T-cell receptors; surface membraneproteins; decay accelerating factor; viral antigens; transport proteins;homing receptors; addressing; regulatory proteins; integrins; tumorassociated antigens; and fragments and thereof.
 28. The method of claim1 or 2, further comprising the step of incorporating the isolatedprotein into a pharmaceutical formulation.